TW202246357A - Bifunctional homodimeric anti-pd-1 and il-15/il-15rα fusion proteins and uses thereof - Google Patents

Abstract

The present disclosure provides bifunctional, homodimeric fusion proteins comprising an anti-PD-1 binding domain component and IL-15/IL-15Rα component, compositions comprising the bifunctional, homodimeric fusion proteins, and methods of treating cancer or viral infection using the bifunctional, homodimeric fusion proteins.

Description

Bifunctional homodimeric anti-PD-1 and IL-15/IL-15Rα fusion protein and its use

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PD-1 antibodies enhance T cell activation by blocking the co-inhibitory PD-1/PD-L1 pathway. PD-1 blockade therapy has revolutionized cancer immunotherapy. Several antagonistic antibodies targeting the PD-1/PD-L1 pathway have been approved by the FDA for the treatment of various cancer indications, including nivolumab (nivolumab, Bristol-Myers Squibb) and pembrolizumab (Merck), Cemiplimab-rwlc (Regeneron), atezolizumab (Roche), durvalumab (AstraZeneca), and avelumab (avelumab, Merck Serono). However, only a fraction of cancer patients respond to PD-1/PD-L1 blockade therapy. Most patients remain refractory to, or develop resistance to, this therapy.

In one aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus : anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, IL-15RαSushi domain, second linker, IL-15 polypeptide, hinge region, and Fc region; where VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment , and the Fc region does not contain a heterodimerization variant mutation.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Comprising: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region ( VH), CH1 domain, first linker, IL-15 polypeptide, second linker, IL-15RαSushi domain, hinge region, and Fc region; where VL, CL, VH and CH1 together form anti-PD-1 Fab fragment, and the Fc region does not contain heterodimerization variant mutations.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), a CH1 domain, a first linker, a modified IL-15RαSushi domain comprising a S40C mutation, a hinge region, and an Fc region; and (c) a modified IL-15 polypeptide comprising a L52C mutation; wherein VL, CL, VH, and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15Rα Sushi domain; and the Fc region does not contain heterodimerization variant mutations.

In yet another aspect, disclosed herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, modified IL-15 polypeptide comprising L52C mutation, hinge region, and Fc region; and (c) modified IL-15RαSushi domain comprising S40C mutation; wherein VL, CL, VH, and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15Rα Sushi domain; and the Fc region does not contain heterodimerization variant mutations.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, IL-15 polypeptide, hinge region, and Fc region; and (c) IL-15RαSushi domain; wherein VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; The IL-15 polypeptide binds to the IL15Rα Sushi domain; and the Fc region does not contain heterodimerization variant mutations.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Comprising: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region ( VH), CH1 domain, first linker, IL-15RαSushi domain, second linker, IL-15 polypeptide, hinge region, and modified Fc region; where VL, CL, VH and CH1 together form an anti-PD -1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Comprising: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region ( VH), CH1 domain, first linker, IL-15 polypeptide, second linker, IL-15RαSushi domain, hinge region, and modified Fc region; where VL, CL, VH and CH1 together form an anti-PD -1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, IL-15RαSushi domain, hinge region, and modified Fc region; and (c) IL-15 polypeptide; wherein VL, CL, VH and CH1 together form an anti-PD-1 The Fab fragment; the IL-15 polypeptide binds to the IL15Rα Sushi domain; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, IL-15 polypeptide, hinge region, and modified Fc region; and (c) IL-15RαSushi domain; wherein VL, CL, VH and CH1 together form an anti-PD-1 The Fab fragment; the IL-15 polypeptide binds to the IL15Rα Sushi domain; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), a CH1 domain, a first linker, a modified IL-15RαSushi domain comprising the S40C mutation, a hinge region, and a modified Fc region; and (c) a modified IL-15 polypeptide comprising the L52C mutation; wherein VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15RαSushi domain; and the modified Fc region contains one or more A mutation selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S.

In yet another aspect, disclosed herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), a CH1 domain, a first linker, a modified IL-15 polypeptide comprising the L52C mutation, a hinge region, and a modified Fc region; and (c) a modified IL-15RαSushi domain comprising the S40C mutation; wherein VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15RαSushi domain; and the modified Fc region contains one or more A mutation selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S.

In another aspect, pharmaceutical compositions comprising the bifunctional homodimeric fusion proteins disclosed herein are provided.

In another aspect, isolated nucleic acid compositions encoding the bifunctional homodimeric fusion proteins disclosed herein are provided.

In another aspect, an expression vector comprising a nucleic acid composition encoding a bifunctional homodimeric fusion protein disclosed herein is provided.

In another aspect, a host cell comprising a nucleic acid composition or expression vector disclosed herein is provided.

In yet another aspect, methods of using the bifunctional homodimeric fusion proteins disclosed herein to inhibit tumor cells, stimulate an immune response, or treat cancer are provided.

Disclosed herein is a bifunctional homodimeric fusion protein comprising an anti-PD-1 binding domain component and an IL-15/IL-15Rα component for stimulating immune responses, treating cancer (optionally in combination with other anti-PD-1 cancer therapeutics), or to treat viral infections (optionally in combination with antiviral agents).

The present disclosure provides a series of bifunctional homodimeric fusion proteins comprising an anti-PD-1 antagonist antibody component and an IL-15/IL-15Rα component to overcome the shortcomings of anti-PD-1 antibody therapy. The bifunctional homodimeric fusion protein disclosed herein has one or more of the following characteristics/advantages: the ability to enhance T cell activation; the ability to promote T cell proliferation and expansion; the ability to promote NK cell activation; and scFv Enhanced stability of the Fab anti-PD1 binding domain compared to the anti-PD1 binding domain; and relative ease of expression and/or purification due to the presence of one or more IgG1 components.

This paper reveals the hypothesis that by increasing the number of tumor infiltrating lymphocytes using IL-15 in combination with anti-PD-1 therapy, antitumor efficacy can be enhanced in those cancer patients. Without being bound by any theory, the bifunctional (inhibition of PD1/PDL1 signaling and enhancement of IL-15 signaling) homodimeric fusion proteins disclosed herein can enhance anti-tumor activity by increasing the number of tumor-infiltrating lymphocytes. Efficacy of tumor therapy. The anti-PD-1 component of the bifunctional homodimeric fusion protein disclosed here enhances T cell activation by removing the co-inhibitory effect of the PD-1/PD-L1 pathway. The IL-15/IL-15Rα complex of the bifunctional homodimeric fusion protein disclosed herein promotes T cell proliferation and expansion to increase circulating T cells and their infiltration in the tumor microenvironment. IL-15 signaling may also have a strong effect on the NK cell population to attack tumor cells. Combinatorial effects were obtained by constructing bifunctional homodimeric fusion proteins comprising anti-PD-1 components and IL-15/IL-15Rα complexes. Specific embodiments of the bifunctional homodimeric fusion proteins disclosed herein retain strong anti-PD1 binding activity, enhanced T cell activation, enhanced T cell proliferation and expansion, enhanced NK cell activation, proliferation and cellular toxicity, and/or demonstrate in vivo antitumor efficacy in mouse tumor models.

Before setting forth the disclosure herein in more detail, it may be helpful to provide definitions of certain terms used herein to understand the same. Other definitions are set forth in the disclosure herein.

In this specification, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range and, where appropriate, fractions thereof (such as tenths of integers). one and one hundredth) or subranges.

As used herein, unless otherwise indicated, the word "about" means ±20% of the stated range, value or structure.

It should be understood that the words "a" and "an" as used herein refer to "one or more" of the listed components. The use of an alternative (eg, "or") should be understood to mean one, both, or any combination of the alternatives.

As used herein, the words "comprising", "having" and "comprising" are used synonymously, and these words and variations thereof are intended to be construed as non-limiting.

"Optional" or "optionally" means that the subsequently described element, component, event or circumstance may or may not occur, and that the description includes the circumstances in which the element, component, event or circumstance occurs and the circumstances in which each other Wait for what doesn't happen.

As used herein, the term "antibody" refers to an intact antibody comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, and has or retains binding to an antigen recognized by the intact antibody. Any antigen-binding portion or fragment of an intact antibody capable of targeting a molecule, such as a scFv, Fab or Fab'2 fragment. Accordingly, the term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab' )2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv) and single domain antibodies (eg sdAb, sdFv, nanobodies). The term includes genetically engineered and/or other modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific, e.g. bispecific Antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA and IgD. Monoclonal antibodies or antigen-binding portions thereof may be non-human, chimeric, humanized or human. The structure and function of immunoglobulins are reviewed, eg, in Harlow et al. eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

The terms "VL" and "VH" refer to the variable binding region from an antibody light chain and an antibody heavy chain, respectively. The variable binding regions comprise discrete, well-defined subregions called "complementarity determining regions" (CDRs) and "framework regions" (FRs). The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable" or "HVR" and refer to the amino acid sequences within the variable region of an antibody that generally confer Antigen specificity and/or binding affinity, wherein consecutive CDRs (ie, CDR1 and CDR2, CDR2 and CDR3) are separated from each other by framework regions in the primary amino acid sequence. There are three CDRs in each variable region (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also called CDRH and CDRL, respectively). In certain embodiments, the antibody VH comprises the following four FRs and three CDRs: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and the antibody VL comprises the following four FRs and three CDRs: FR1-LCDR1 -FR2-LCDR2-FR3-LCDR3-FR4. In general, VH and VL together form an antigen-binding site through their respective CDRs.

Numbering of CDRs and framework regions can be determined according to any known method or scheme, such as the Kabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g., Kabat et al.: "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, Fifth Edition; Chothia and Lesk et al: J. Mol. Biol , 196:901–917 (1987); Lefranc et al: Dev. Comp , Immunol. 27:55, 2003; Honegger and Plückthun: J. Mol. Bio , 309:657–670 (2001)). The Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298–300) can be used to annotate equivalent residue positions and compare equivalent residue positions across different molecules. Accordingly, identification of the CDRs of the exemplary variable domain (VH or VL) sequences provided herein according to one numbering scheme does not exclude antibodies comprising CDRs of the same variable domain determined using a different numbering scheme. Unless otherwise stated, the CDRs of the anti-PD-1 antibodies provided in the disclosure herein are identified according to the IMGT numbering scheme.

As used herein, "Fab" or "Fab fragment" or "fragment antigen binding" refers to the antigen binding region of an antibody, which consists of a constant domain and a variable domain of each of the heavy and light chains (VH-CH1 and VL-CL). Composed of variable regions. Fab fragments can be produced by enzymatic digestion of full-length antibodies, chemical digestion of full-length antibodies, or recombinant synthesis.

As used herein, "Fc region" refers to a fragment of the heavy chain constant region from the Fc fragment of an antibody (a "fragment crystallizable" region or Fc region), which may include one or more constant domains of the antibody, such as CH2, CH3 or Both, and not including the CH1 domain.

As used herein, "PD-1" or "programmed cell death protein 1" or "CD279" refers to an immune checkpoint molecule expressed on the surface of T cells, B cells, and macrophages, which acts by promoting The apoptosis of specific T cells and the reduction of apoptosis of regulatory T cells play a role in the negative regulation of the immune system. PD-1 is a member of the CD28/CTLA-4 family of type I membrane proteins and T cell regulators. The PD-1 protein consists of an extracellular IgV domain, followed by a transmembrane region and an intracellular region. The intracellular region contains two phosphorylation sites located on the immunoreceptor tyrosine inhibitor motif and the immunoreceptor tyrosine switch motif. PD-1 binds to two ligands, PD-L1 and PD-L2. PD-1 includes mammalian PD-1 proteins, such as humans and non-human primates. In some specific embodiments, PD-1 is human PD-1 (NCBI reference sequence NP_005009; SEQ ID NO: 15) or macaque PD-1 (Gene Bank accession number ABR15751; SEQ ID NO: 16).

As used herein, "IL-15" or "Interleukin-15" refers to a pro-inflammatory cytokine that stimulates the activation, proliferation and cytolytic activity of CD8 T cells and natural killer (NK) cells. IL-15 occurs in two forms: (1) soluble IL-15 (IL-15sol) and (2) complexed with its proprietary receptor IL-15Rα, forming the IL-15 receptor complex (IL-15Rc). IL-15Rc is transduced to neighboring cells expressing IL-15Rβ/γ, exerting enhanced biological activity compared to IL-15sol alone. In some embodiments, IL-15 relates to a form of IL-15 capable of complexing with IL-15Rα. In some embodiments, the IL-15 relates to human IL-15, such as human IL-15 comprising the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the IL-15 relates to a modified IL-15 containing one or more mutations. In some specific embodiments, the modified IL-15 comprises a L52C substitution (refer to SEQ ID NO:20 for amino acid numbering), such as the modified IL-15 comprising the amino acid sequence shown in SEQ ID NO:22.

As used herein, "IL-15Rα Sushi domain" or "Sushi domain" or "Sushi" refers to the shortest part of IL-15 receptor α that retains IL-15 binding activity. Soluble IL-15Rα Sushi domain is a 65 amino acid peptide comprising the extracellular Sushi domain of IL-15Rα. The Sushi domain is a common motif in protein-protein interactions, which contains four cysteines, forming two disulfide bonds in 1-3 and 2-4 patterns. In some embodiments, the IL-15RαSushi domain relates to a human IL-15RαSushi domain, such as a human IL-15RαSushi domain comprising the amino acid sequence set forth in SEQ ID NO:21. IL-15Rα Sushi domains include modified IL-15Rα Sushi domains containing one or more mutations. In some embodiments, the modified IL-15RαSushi domain comprises an S40 substitution (refer to SEQ ID NO:21 for amino acid numbering), such as the modified IL-15RαSushi domain comprising the amine shown in SEQ ID NO:23 amino acid sequence.

As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, that is, the α-carbon bonded to hydrogen, carboxyl, amine and R groups, such as homoserine, norleucine, methylthio Amino acid sulfide, methionine methyl sulfide. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that are structurally different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or a polypeptide molecule compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can result in several different types of sequence changes, including nucleotide or amino acid substitutions, insertions or deletions.

As used herein, "modification" may refer to a mutation in a nucleic acid or polypeptide sequence, or an alteration to a moiety chemically linked to a protein, compared to a reference or wild-type nucleic acid or polypeptide molecule, respectively. For example, a modification can be an altered carbohydrate or PEG structure attached to the protein.

As used herein, a "protein" or "polypeptide" as used herein refers to a compound consisting of amino acid residues covalently linked by peptide bonds. The term "protein" may be synonymous with the term "polypeptide", or may otherwise refer to a complex of two or more polypeptides. Polypeptides may further contain other components (eg, covalently bound), such as tags, labels, biologically active molecules, or any combination thereof. In certain embodiments, polypeptides may be fragments. As used herein, "fragment" means a polypeptide lacking one or more amino acids found in a reference sequence. A fragment may comprise a binding domain, antigen or epitope found in the reference sequence. A fragment of a reference polypeptide may have at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% of the amino acid sequence of the reference sequence %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more amino acids.

As described herein, a "variant" polypeptide species has one or more unnatural amino acids, one or more amino acid substitutions, one or more Amino acid insertions, deletions of one or more amino acids, or any combination thereof. In certain embodiments, "variant" refers to a polypeptide having substantially similar activity (eg, enzyme function, immunogenicity, specific binding activity) or structure relative to a reference polypeptide. A variant of a reference polypeptide may have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity, as determined by sequence alignment programs and parameters known in the art of. Variants may arise, for example, from genetic polymorphisms or human manipulation. Conservative substitutions of amino acids are well known and may occur naturally or may be introduced during recombinant production of the protein. Amino acid substitutions, deletions, and additions to the introduced protein can be made using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning : A Laboratory Manual , 3d ed., Cold Spring Harbor Laboratory Press, NY, 2001 ). Oligonucleotide-directed site-specific (or segment-specific) mutagenesis methods can be employed to provide altered polynucleotides with specific codons altered according to desired substitutions, deletions or insertions. Alternatively, random or saturation mutagenesis techniques such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis can be used to generate polypeptide variants (see, eg, Sambrook et al., supra).

"Conservative substitutions" relate to amino acid substitutions that do not significantly affect or alter the binding characteristics of a particular protein. In general, a conservative substitution is one in which the amino acid residue being substituted is replaced by an amino acid residue having a similar side chain. Conservative substitutions include those found in the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T) ; group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); group 3: asparagine (Asn or N), glutamic acid (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), methionine (Met or M), valine (Val or V); and group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Alternatively, or rather, amino acids may be classified as conservative substituents by similar function, chemical structure or composition (eg, acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, for substitution purposes, aliphatic groups can include Gly, Ala, Val, Leu, and Ile. Other conservative substituents include: sulfur-containing: Met and cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic nonpolar residues: Met , Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

In the context of two or more polypeptide or nucleic acid molecule sequences, when comparing and aligning for maximum correspondence over a comparison window or specific region, such as using methods known in the art (such as sequence comparison algorithms), by The terms "identical" or "percent identity", as measured by manual alignment or by visual inspection, mean that two or more sequences or subsequences are identical, or have a specified percentage of amino acid residues that are identical over specified regions bases or nucleotides (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% or 100% identity). The algorithm used in this paper to determine the percentage of sequence identity and sequence similarity is the BLAST 2.0 algorithm, such as Altschul et al. "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 2007, 25, 3389 – as described in 3402, where the parameter is set to the default value.

As used herein, a "fusion protein" comprises a single-chain polypeptide having at least two distinct domains, where the domains do not naturally occur together in the protein. Nucleic acid molecules encoding fusion proteins can be constructed using PCR, recombinant engineering, etc., or such fusion proteins can be prepared synthetically. Fusion proteins may further contain (eg, covalently bond) other components, such as tags, linkers, or biologically active molecules.

"Nucleic acid molecule" or "polynucleotide" refers to a polymeric compound comprising nucleotides covalently linked by 3'-5' phosphodiester bonds. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes genomic DNA, mitochondrial DNA, cDNA, or vector DNA. Nucleic acid molecules can be double-stranded or single-stranded, and if single-stranded, can be coding strands or non-coding (antisense strands). Nucleic acid molecules may contain natural or non-natural subunits. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences encoding the same amino acid sequence. Some versions of the nucleotide sequence may also include introns such that introns can be removed by co-transcriptional or post-transcriptional machinery. In other words, due to the redundancy or degeneracy of the genetic code, or through splicing, different nucleotide sequences can encode the same amino acid sequence.

Variants of the polynucleotides disclosed herein are also contemplated. A variant polynucleotide is at least 80%, 85%, 90%, 95%, 99%, or 99.9% identical to a reference polynucleotide described herein, or 0.015 M sodium chloride, 0.0015 M Sodium citrate or the stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide at about 42° C. hybridize to the reference polynucleotide of defined sequence. The polynucleotide variant retains the ability to encode an immunoglobulin-like binding protein or antigen-binding fragment thereof having the functionality described herein.

The term "isolated" means that the material has been removed from its original environment (eg, the natural environment if the original environment occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, whereas the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides can be part of a vector and/or such polynucleotides or polypeptides can be part of a composition (e.g. a cell lysate) and still be isolated because such vectors or compositions are not nucleic acids or part of the natural environment of the polypeptide.

As used herein, the terms "modified", "recombinant" or "non-natural" refer to organisms, microorganisms, cells, Nucleic acid molecules or vectors in which such alterations or modifications are introduced by genetic modification (ie, human intervention). Genetic alterations include, for example: modifications introducing expressible nucleic acid molecules encoding functional RNAs, proteins, fusion proteins or enzymes; or additions, deletions, substitutions of other nucleic acid molecules; or other functional disruptions of cellular genetic material. Other modifications include, for example, non-coding regulatory regions, where such modifications alter the expression of a polynucleotide, gene or operon.

As used herein, a "heterologous" or "exogenous" nucleic acid molecule, construct or sequence refers to a nucleic acid molecule, construct or sequence that is not native to the host cell, but may be homologous to a nucleic acid molecule or a portion of a nucleic acid molecule from the host cell. The source of a heterologous or exogenous nucleic acid molecule, construct or sequence may be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule is added (i.e., non-endogenous or native) to the host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, etc., wherein the added Molecules can integrate into the host genome or exist as extrachromosomal genetic material (for example, as plastids or other forms of self-replicating vectors), possibly in multiple copies. Furthermore, "heterologous" relates to a non-native enzyme, protein or other activity encoded by a foreign nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity.

As used herein, the words "endogenous" or "native" relate to a gene, protein or activity normally present in the host cell. Furthermore, a gene, protein or activity that is mutated, overrepresented, shuffled, duplicated or otherwise altered compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell. For example, endogenous control sequences (e.g., promoters, translation-inducing sequences) from a first gene can be used to alter or regulate the expression of a second native gene or nucleic acid molecule, where the second native gene or nucleic acid molecule is expressed or regulated differently than the parent Normal expression or regulation in this cell.

As used herein, the term "expression" refers to the process of producing a polypeptide based on the coding sequence of a nucleic acid molecule, such as a gene. A process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (eg, a promoter).

As described herein, more than one heterologous nucleic acid molecule can be present as a single nucleic acid molecule, as multiple individually controlled genes, as a polycistronic nucleic acid molecule (such as the heavy and light chains of an antibody), as a protein-encoding Single nucleic acid molecules (eg, heavy chains of antibodies), or any combination thereof, are introduced into the host cell. When two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be present as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, Introduced by integration into the host chromosome at a single site or multiple sites, or any combination thereof. References to the number of heterologous nucleic acid molecules or protein activities refer to the number of encoding nucleic acid molecules or protein activities, not the number of individual nucleic acid molecules introduced into the host cell.

As used herein, in the context of inserting a nucleic acid sequence into a cell, the term "introducing" means "transfecting" or "transformation" or "transduction" and includes references to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell, Among them, nucleic acid molecules can be incorporated into the genome of cells (such as chromosomes, plastids, chromosomal or mitochondrial DNA), transformed into autonomous replicons, or expressed transiently (such as transfected mRNA).

The following sections provide additional definitions. I. Anti- PD1 component

The bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein comprises an anti-PD-1 binding domain. In some embodiments, the anti-PD-1 binding domain comprises a Fab fragment. Fab fragments consist of two polypeptide chains, each comprising a constant domain and a variable region of each heavy and light chain. The first polypeptide chain includes from N-terminal to C-terminal: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL). The second polypeptide chain includes from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH) and heavy chain constant domain 1 (CH1). The VL-CL and VH-CH1 of the two polypeptide chains together form the anti-PD-1 Fab fragment. In some embodiments, the anti-PD1 Fab fragment formed by the first polypeptide chain and the second polypeptide chain contains a cysteine residue in the CL domain and a cysteine residue in the CH1 domain Interchain disulfide bonds between groups. In some other specific embodiments, the anti-PD1 Fab fragment formed by the first polypeptide chain and the second polypeptide chain contains two or more cysteine residues in the CL domain and CH1 domain Interchain disulfide bonds between cysteine residues.

In some embodiments, the anti-PD-1 Fab fragment is chimeric, human or humanized.

In some embodiments, the anti-PD-1 Fab fragment is IgG1, IgG2, IgG3 or IgG4 isotype.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL and CH1 domains are IgG1, IgG2, IgG3 or IgG4 isotype. In general, the CH1 domain relates to positions 118 to 220 according to the EU index as in Kabat. In some embodiments, CL comprises a CK constant region. In some embodiments, CL comprises a C lambda constant region. In some embodiments, the CL domain is an IgG1 CK domain comprising the amino acid sequence shown in SEQ ID NO:12. In some embodiments, the CH1 domain is an IgG1 CH1 domain comprising the amino acid sequence shown in SEQ ID NO:11.

In some specific embodiments, the first polypeptide chain comprising the light chain variable region (VL) and the light chain constant region (CL) of the anti-PD1 antibody comprises the amino acid sequence shown in SEQ ID NO:34. In some specific embodiments, the anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL) are encoded by the nucleic acid sequence shown in SEQ ID NO:45.

In some specific embodiments, the second polypeptide chain comprising the heavy chain variable region (VH) of the anti-PD1 antibody and the heavy chain constant domain 1 (CH1) comprises the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 35 Amino acids 21 to 236 of ID NO:35. In some specific embodiments, the anti-PD1 antibody heavy chain variable region (VH) and heavy chain constant domain 1 (CH1) are encoded by the nucleic acid sequence shown in SEQ ID NO:36.

In some specific embodiments, the first polypeptide chain comprising the light chain variable region (VL) and the light chain constant region (CL) of an anti-PD1 antibody comprises the amino acid sequence shown in SEQ ID No: 34; and comprises an anti-PD1 antibody The second polypeptide chain of the heavy chain variable region (VH) of the PD1 antibody and the heavy chain constant domain 1 (CH1) comprises the amino acid sequence shown in SEQ ID NO:35 or amino acid 21 of SEQ ID NO:35 to 236. II , IL-15/IL-15RαSushi domain components

The bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein comprises an IL-15 polypeptide, an IL-15Rα Sushi domain, or an IL-15 polypeptide and IL-15 within the second polypeptide chain. Both of the 15RαSushi domains, the second polypeptide chain was covalently linked to the C-terminus of the VH-CH1 of the anti-PD-1 Fab fragment using a first linker. In some embodiments, the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein comprises an IL-15 polypeptide within a second polypeptide chain using The first linker is covalently linked to the C-terminus of VH-CH1 of the anti-PD-1 Fab fragment, and the IL-15Rα Sushi domain is contained in a separate third polypeptide chain. In some embodiments, the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein comprises an IL-15Rα Sushi domain within a second polypeptide chain that The first linker is used to covalently connect to the C-terminus of the VH-CH1 of the anti-PD-1 Fab fragment, and the IL-15 polypeptide is contained in a separate third polypeptide chain. In some specific embodiments, the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein comprises an IL-15Rα Sushi domain and an IL-15 polypeptide within the second polypeptide chain, wherein The IL-15RαSushi domain or IL-15 polypeptide is connected to the C-terminal of the VH-CH1 of the anti-PD-1 Fab fragment using the first linker.

The bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein uses the Sushi domain of IL-15Rα to complex with the IL-15 polypeptide within the same polypeptide chain or on a separate polypeptide chain.

In some embodiments, the IL-15 polypeptide is a human IL-15 polypeptide sequence. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:20. In some embodiments, the IL-15 polypeptide is encoded by the nucleic acid sequence shown in SEQ ID NO:42.

In some embodiments, the IL-15Rα Sushi domain is a human IL-15 Sushi domain. In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:21. In some specific embodiments, the IL-15Rα Sushi domain is encoded by the nucleic acid sequence shown in SEQ ID NO:40.

In some embodiments, the IL-15 polypeptide is a modified IL-15 polypeptide. In some embodiments, the modified IL-15 polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO:20 %, 96%, 97%, 98%, or 99% identity. In some embodiments, compared to the amino acid sequence shown in SEQ ID NO: 20, the modified IL-15 polypeptide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or more amino acid mutations (eg substitutions, insertions and/or deletions). In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the IL-15Rα Sushi domain is a modified IL-15Rα Sushi domain. In some specific embodiments, the IL-15Rα Sushi domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, compared to the amino acid sequence shown in SEQ ID NO: 21, the modified IL-15Rα Sushi domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acid mutations (eg substitutions, insertions and/or deletions). In some embodiments, the amino acid substitutions are conservative amino acid substitutions.

In some embodiments, the modified IL-15 polypeptide comprises the N72D mutation. The N72D mutation increases the binding affinity of IL-15 to the IL-2/15Rβγc complex and significantly enhances the biological activity of IL-15.

In some embodiments, the modified IL-15 polypeptide comprises an I67E mutation. (Amino acid positions refer to SEQ ID NO:20). In some embodiments, the modified IL-15 I67E polypeptide comprises the amino acid sequence of SEQ ID NO:52. In some specific embodiments, the IL-15 I67E polypeptide is encoded by the nucleic acid sequence shown in SEQ ID NO:53.

In some embodiments, each of the IL-15 polypeptide and the IL-15RαSushi domain is engineered to contain a cysteine residue that forms a disulfide bond between the IL-15 polypeptide and the IL-15RαSushi domain. In some embodiments, the modified IL-15R polypeptide comprises a substitution selected from L45C, Q48C, V49C, L52C, E53C, E87C, E89C or any combination thereof (for amino acid positions, refer to SEQ ID NO: 20). In some specific embodiments, the modified IL-15 polypeptide comprises an L52C amino acid substitution (refer to SEQ ID NO: 20 for the amino acid position). In some embodiments, the modified IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the modified IL-15 polypeptide is encoded by the nucleic acid sequence shown in SEQ ID NO:43. In some specific embodiments, the modified IL-15Rα Sushi domain comprises substitutions selected from K34C, A37C, G38C, S40C, L42C or any combination thereof (for amino acid positions, refer to SEQ ID NO: 21). In some specific embodiments, the IL-15RαSushi domain comprises a S40C substitution (refer to SEQ ID NO: 21 for the amino acid position). In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:23. In some specific embodiments, the IL-15Rα Sushi domain is encoded by the nucleic acid sequence shown in SEQ ID NO:41. In some embodiments, the modified IL-15 polypeptide and the modified IL-15Rα Sushi domain in the bifunctional homodimer fusion protein respectively comprise an amino acid substitution group selected from: L52C:S40C; V49C:S40C; E89C:K34C; Q48C:G38C; E53C:L42C; and L45C:A37C. In some embodiments, the modified IL-15 polypeptide and the modified IL-15Rα Sushi domain in the bifunctional homodimeric fusion protein respectively comprise an amino acid substitution group of L52C:S40C. III , Fc region

The bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein comprises an Fc region. The Fc region is responsible for the effector functions of the antibody, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors (eg, CD16, CD32, FcRn), relative to Longer in vivo half-life, protein A binding, and possibly even placental transfer of polypeptides lacking the Fc region (see Capon et al.: Nature 337:525, 1989). In some embodiments, the Fc region comprises the CH2 and CH3 domains of an IgG antibody. In some embodiments, the Fc region is obtained from a human antibody. In some embodiments, the Fc region comprises CH2 and CH3 domains from the same antibody isotype, such as human IgGl, IgG2, IgG3 or IgG4 (eg CH2CH3 from human IgGl).

In some embodiments, the Fc region of the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion proteins disclosed herein will be capable of mediating one or more of these effector functions. In some embodiments, the Fc region has normal effector function, meaning that it has less than 25%, 20%, less than 25%, 20%, or less effector function (e.g., ADCC, CDC, or both) compared to a wild-type antibody of the same isotype. 15%, 10%, 5%, 1% difference.

In some embodiments, the Fc region of the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein is activated by, for example, one or more amines in the Fc region portion known in the art. Amino acid substitution or deletion with a reduction in one or more of these effector functions or lack of one or more effector functions. An antibody or antigen-binding fragment having a modified, mutated or variant Fc region with reduced effector function means that the antibody exhibits FcR binding, ADCC, CDC, or any of these, compared to an antibody having a wild-type Fc region portion of the same isotype. Combined reduction of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. In some embodiments, the mutated or variant Fc region exhibits reduced binding to FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD16a), FcyRIIIB (CD16b), or any combination thereof. In some embodiments, the Fc region in an antibody or antigen-binding fragment disclosed herein is a modified Fc region with reduced ADCC, CDC, or both reduced ADCC and CDC.

In some embodiments, the Fc region is a modified Fc region comprising one, two, three, Four, five, six or all.

In some embodiments, the Fc region is a modified IgG1 Fc region comprising mutations corresponding to E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S according to EU numbering as set forth in Kabat to attenuate Fc-mediated Effector functions (ADCC and CDC). In some embodiments, the modified Fc region comprises the amino acid sequence shown in SEQ ID NO:19. In some embodiments, the modified Fc region is encoded by the nucleic acid sequence shown in SEQ ID NO:44.

In some embodiments, the Fc region does not comprise heterodimerization variant mutations. A "heterodimerization variant mutation" involves a mutation in the amino acid sequence of each Fc region on two polypeptide chains that promotes heterodimer formation of the two polypeptide chains and/or makes the heterodimer ratio identical Source dimers are easier to purify. Heterodimerization variant mutations may comprise steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof.

"Steric mutation" involves amino acid mutations in each Fc region-containing polypeptide chain that allow heterodimers to associate more readily than homodimers. Space mutations include knob and hole mutations and charge pair mutations.

"Knob-hole mutation", also known as "skew mutation", involves amino acid modification in the CH3 domain to create a "knob" or "hole" in each heavy chain to promote heterodimerization (please See, eg, Ridgway et al., Protein Engineering (1996) 9:617; Atwell et al., J. Mol. Biol. (1997) 270:26; and US Patent No. 8,216,805). Knob-hole mutations create complementary interaction interfaces by manipulating key amino acid residues involved in Fc dimer interactions. Amino acids with small side chains are replaced by amino acids with large side chains, creating knobs or protrusions in one polypeptide chain, and vice versa, creating sockets or sockets in the paired polypeptide chain. In some embodiments, the knob-hole mutation comprises T366Y paired with a Y407T mutation. Other exemplary knob-hole mutations include S364K/E357Q paired with L368D/370S; L368D/K370S paired with S364K; L368E/K370S paired with S364K; T411T/E360E/Q362E paired with D401K; L368D/370S; K370S paired with S364K/E357Q; and T366S/L368A/Y407V paired with T366W (optionally including bridging disulfides, T366S/L368A/Y407V/Y349C paired with T366W/S354C).

"Electrostatic steering mutations" also known as "charge pair mutations" involve amino acid mutations that use electrostatics to bias the formation of heterodimers in favor of homodimers (see, e.g., Gunasekaran et al., J. Biol. Chem.( 2010) 285:19637). Electrostatic steering mutations generate altered charge polarity at the Fc-dimer interface such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions that promote the desired Fc heterodimer formation versus unfavorable repulsive charge interactions Inhibits unwanted Fc homodimer formation. Exemplary electrostatic steering mutations include: D221E/P228E/L368E paired with D221R/P228R/K409R, and C220E/P228E/L368E paired with C220R/E224R/P228R/K409R.

"pi mutation" involves an amino acid mutation in the Fc region of one or both polypeptide chains, which is intended to alter the isoelectric point (pi) of one or both of the Fc-containing polypeptide chains so that heterodimers can Separation from homodimers. The pi of one Fc-region-containing polypeptide chain is engineered away from the pi of a second Fc-region-containing polypeptide chain, or both Fc-containing polypeptide chains can be modified so that the pi of one polypeptide chain is increased and the pi of the other polypeptide chain is increased. pi decreases. A change in pi can be achieved by removing or adding a charged residue, changing a charged residue from a positive or negative charge to the opposite charge, or changing a charged residue to a neutral residue. The number of pi variants required to obtain good separation of heterodimers from homodimers on one or both polypeptide chains depends in part on the starting pi of the polypeptide chains. In general, separation of heterodimers from homodimers can be achieved if the pi values of the two polypeptide chains differ by at least 0.1 pH units, or preferably at least 0.2, 0.3, 0.4 or 0.5 pH units.

According to the EU index, exemplary heterodimerization variant mutations include: S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L ；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/K370S:S364K/E357L；D221E/P228E/L368E:D221R /P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is a modified IgG1 Fc region comprising mutations corresponding to E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S according to EU numbering as set forth in Kabat, and does not comprise iso Dimerization variant mutation. In some embodiments, the modified Fc region comprises the amino acid sequence shown in SEQ ID NO: 19 and does not comprise heterodimerization variant mutations. IV , hinge area

The bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein comprises a hinge region. Antibodies have a hinge sequence generally located between the Fab and Fc regions (although the lower portion of the hinge may include the amino-terminal portion of the Fc region). As background, the immunoglobulin hinge acts as a flexible spacer to allow the Fab moiety to move freely in space. In contrast to the constant regions, hinges are structurally diverse, varying in sequence and length between immunoglobulin classes and even subclasses. The antibody hinge region can be divided into three regions: the upper hinge, the middle (or core) hinge, and the lower hinge, each of which has a different functional role. The upper hinge allows the Fab to move and rotate. The central core hinge contains a variable number of cysteine residues, depending on the IgG subtype that forms the disulfide bond, to stabilize the association of the antibody heavy chain. On the C-terminal side is the lower hinge that allows Fc to move relative to Fab, and its amino acid residues can participate in FcγR binding. The human IgG1 hinge region consists of 23 amino acids and has free flexibility, which allows the Fab fragment to rotate about its axis of symmetry and move within a sphere centered on the first of the two interheavy-chain disulfide bonds . In contrast, the human IgG2 hinge is relatively short (19 amino acids) and contains a rigid polyproline double helix stabilized by four interheavy chain disulfide bonds, which limits flexibility. The human IgG3 hinge differs from other subclasses by its unique extended hinge region (approximately four times as long as the IgG1 hinge), which contains 62 amino acids (including 21 prolines and 11 cysteines ), forming a non-flexible polyproline duplex and providing greater flexibility because the Fab fragment is relatively far away from the Fc fragment. The hinge length of human IgG4 is 20 amino acids, shorter than IgG1, but similar in length to IgG2 and has two disulfide bonds between heavy chains, and its flexibility is between IgG1 and IgG2. The structure and function of immunoglobulins are reviewed, eg, in Harlow et al. eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

In the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein, the hinge region connects the C-terminus of the IL-15 polypeptide or the IL-15Rα Sushi domain with the Fc in the second polypeptide chain. The N-terminal link of the region.

In some embodiments, the length of the hinge region is about 10 to about 100 amino acids, about 10 to about 90 amino acids, about 10 to about 80 amino acids, about 10 to about 70 amino acids, about 10 to about 60 amino acids, about 10 to about 50 amino acids, about 10 to about 40 amino acids, about 10 to about 30 amino acids, From about 10 to about 25 amino acids, or from about 10 to about 20 amino acids.

In some embodiments, the bifunctional homodimeric fusion protein forms at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more disulfide bonds.

In some embodiments, the hinge region is a modified hinge region that is at least 70%, 75%, 80%, 85%, 90% identical to a reference hinge region such as IgG1 hinge-EPKSCDKTHTCPPCP (SEQ ID NO: 56) , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. In some embodiments, the hinge region is a modified hinge region having 1, 2, 3, 4, 5, 6, 7 compared to a reference hinge region such as IgG1 hinge - EPKSCDKTHTCPPCP (SEQ ID NO: 56) , 8, 9, 10, 11, 12, 13, 14, 15 or more mutations (substitutions, insertions, and/or deletions). In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the modified hinge region has altered flexibility. Mutations that increase domain flexibility include substitution of one or more glycine residues, cysteines involved in disulfide bond formation by amino acid residues that cannot form disulfide bonds (e.g., serine, alanine, Glycine) substitution. Mutations that reduce the flexibility of the hinge region include substitution of one or more amino acid residues with one or more proline residues, substitution of amino acid residues capable of forming disulfide bonds (such as cysteine) that cannot form Disulfide-bonded amino acid residues (eg, serine, alanine, glycine).

In some embodiments, the hinge region is a human antibody hinge region. In some embodiments, the hinge region is an IgG1, IgG2, IgG3 or IgG4 hinge region. In some embodiments, the hinge region comprises an upper hinge or portion thereof, a middle (or core) hinge or portion thereof, and a lower hinge or portion thereof. In some embodiments, the upper hinge or portion thereof, the middle hinge or portion thereof, and the lower hinge or portion thereof are each obtained from the same antibody isotype (eg, the upper, middle, and lower hinges are all obtained from IgG1). In some embodiments, the hinge region is a modified hinge region wherein the upper hinge or portion thereof, the middle hinge or portion thereof, and the lower hinge or portion thereof are obtained from two or more antibody isotypes. In some embodiments, the hinge region comprises an upper hinge, or portion thereof, and a middle hinge, or portion thereof. In some embodiments, the upper hinge, or portion thereof, and the middle hinge, or portion thereof, are each obtained from the same antibody isotype (eg, both the upper and middle hinges are derived from IgG1). In some embodiments, the hinge region is a modified hinge region wherein the upper hinge or portion thereof and the middle hinge or portion thereof are obtained from two different antibody isotypes. In some embodiments, the hinge region comprises an upper hinge or a portion thereof obtained from an IgG1 isotype and a middle hinge. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:17. V , linker

The bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein contains one or more linkers connecting various domains in the polypeptide chain. In the second polypeptide chain, the anti-PD-1 heavy chain Fab component (VH-CH1), IL-15 polypeptide and/or IL-15RαSushi domain, and hinge region-Fc region can each be connected to each other by linking peptides. separate. Linkers are amino acid sequences of sufficient length to allow proteins to form appropriate secondary and tertiary structures. In some embodiments, the linker comprises 1 to 30 amino acids, 10 to 30 amino acids, or 15 to 30 amino acids. In some embodiments, the linker is a flexible linker. In some embodiments, the linker does not exhibit a tendency to form secondary structures that can interact with the functional domains of the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein. In some embodiments, the linker has a minimum of hydrophobic or charged residues that facilitate interaction with the functional domain of the fusion protein. In some embodiments, the linker comprises Gly, Asn and/or Ser residues.

In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 24 or 46. In some embodiments, the linker is encoded by the nucleic acid sequence shown in SEQ ID NO:38, 39 or 47.

In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:25. In some embodiments, the linker is encoded by the nucleic acid sequence shown in SEQ ID NO:37.

The bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion proteins disclosed herein may also contain linked amino acids involving one or more (e.g., about 1 to 20) amino acids in the two phases of the polypeptide. Amino acid residues between adjacent motifs, regions or domains. Linking amino acids can be generated by chimeric protein construct design (eg, amino acid residues created using restriction sites during construction of a nucleic acid molecule encoding a fusion protein). VI , bifunctional homodimeric anti- PD1/IL-15/IL-15Rα fusion protein

Disclosed herein provides a bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein comprising two functional components: IL-15/IL bound to IL-15Rβγc complex expressed on the cell surface -15RαSushi component (IL-15 complex), and anti-PD-1 component (Fab) that binds to PD-1. When used in conjunction with the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion proteins disclosed herein, the term "homodimer" means that the fusion protein has at least two associated Fc-containing Regions of polypeptide chains that self-assemble into fusion proteins containing homodimeric Fc regions. Therefore, the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein may comprise more than two polypeptide chains, such as tetrameric (BFP1 and BFP6) or hexameric structures (BFP2 to BFP5), as long as the two polypeptide chains containing the Fc region form a homodimer.

In one aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus : anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, IL-15RαSushi domain, second linker, IL-15 polypeptide, hinge region, and Fc region; where VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment , and the Fc region does not contain a heterodimerization variant mutation. Figure 1A (BFP1) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence shown in SEQ ID NO:14, and VH comprises the amino acid sequence shown in SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker and/or the second linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:20.

In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:21.

In some embodiments, the second polypeptide chain further comprises a third linker between the IL-15 polypeptide and the hinge region. In some embodiments, the third linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:27 or amino acids 21 to 695 of SEQ ID NO:27. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:26 The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Comprising: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region ( VH), CH1 domain, first linker, IL-15RαSushi domain, second linker, IL-15 polypeptide, hinge region, and modified Fc region; where VL, CL, VH and CH1 together form an anti-PD -1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. Figure 1A (BFP1) or Figure 1G (BFP7) depict exemplary bifunctional homodimeric fusion proteins according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker and/or the second linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:20. In some embodiments, the IL-15 polypeptide is a modified IL-15 with an I67E mutation and comprises the amino acid sequence shown in SEQ ID NO:52.

In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:21.

In some embodiments, the second polypeptide chain further comprises a third linker between the IL-15 polypeptide and the hinge region. In some embodiments, the third linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the Fc region does not comprise heterodimerization variant mutations. In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:27 or amino acids 21 to 695 of SEQ ID NO:27. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:26 The nucleic acid sequence shown encodes.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:54 or amino acids 21 to 695 of SEQ ID NO:54. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:55. The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Comprising: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region ( VH), CH1 domain, first linker, IL-15 polypeptide, second linker, IL-15RαSushi domain, hinge region, and Fc region; where VL, CL, VH and CH1 together form anti-PD-1 Fab fragment, and the Fc region does not contain heterodimerization variant mutations. Figure 1F (BFP6) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker and/or the second linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:20.

In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:21.

In some embodiments, the second polypeptide chain further comprises a third linker between the IL-15Rα Sushi domain and the hinge region. In some embodiments, the third linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:10 or amino acids 21 to 695 of SEQ ID NO:10. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:9 The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Comprising: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region ( VH), CH1 domain, first linker, IL-15 polypeptide, second linker, IL-15RαSushi domain, hinge region, and modified Fc region; where VL, CL, VH and CH1 together form an anti-PD -1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. Figure 1F (BFP6) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker and/or the second linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:20.

In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:21.

In some embodiments, the second polypeptide chain further comprises a third linker between the IL-15Rα Sushi domain and the hinge region. In some embodiments, the third linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the Fc region does not comprise heterodimerization variant mutations. In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:10 or amino acids 21 to 695 of SEQ ID NO:10. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:9 The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, IL-15RαSushi domain, hinge region, and modified Fc region; and (c) IL-15 polypeptide; wherein VL, CL, VH and CH1 together form an anti-PD-1 The Fab fragment; the IL-15 polypeptide binds to the IL15Rα Sushi domain; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. Figure 1B (BFP2) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:20.

In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:21.

In some embodiments, the second polypeptide chain further comprises a second linker between the IL-15Rα Sushi domain and the hinge region. In some embodiments, the second linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the Fc region does not comprise heterodimerization variant mutations. In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:29 or amino acids 21 to 561 of SEQ ID NO:29. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:28 The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), a CH1 domain, a first linker, a modified IL-15RαSushi domain comprising a S40C mutation, a hinge region, and an Fc region; and (c) a modified IL-15 polypeptide comprising a L52C mutation; wherein VL, CL, VH, and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15Rα Sushi domain; and the Fc region does not contain heterodimerization variant mutations. Figure 1C (BFP3) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the modified IL-15 polypeptide comprising the L52C mutation comprises the amino acid sequence set forth in SEQ ID NO:22.

In some embodiments, the modified IL-15Rα Sushi domain comprising the S40C mutation comprises the amino acid sequence set forth in SEQ ID NO:23.

In some embodiments, the second polypeptide chain further comprises a second linker between the modified IL-15Rα Sushi domain and the hinge region. In some embodiments, the second linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:31 or amino acids 21 to 561 of SEQ ID NO:31. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:30 The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), a CH1 domain, a first linker, a modified IL-15RαSushi domain comprising the S40C mutation, a hinge region, and a modified Fc region; and (c) a modified IL-15 polypeptide comprising the L52C mutation; wherein VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15RαSushi domain; and the modified Fc region contains one or more A mutation selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. Figure 1C (BFP3) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the modified IL-15 polypeptide comprising the L52C mutation comprises the amino acid sequence set forth in SEQ ID NO:22.

In some embodiments, the modified IL-15Rα Sushi domain comprising the S40C mutation comprises the amino acid sequence set forth in SEQ ID NO:23.

In some embodiments, the second polypeptide chain further comprises a second linker between the modified IL-15Rα Sushi domain and the hinge region. In some embodiments, the second linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the Fc region does not comprise heterodimerization variant mutations. In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:31 or amino acids 21 to 561 of SEQ ID NO:31. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:30 The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, modified IL-15 polypeptide comprising L52C mutation, hinge region, and Fc region; and (c) modified IL-15RαSushi domain comprising S40C mutation; wherein VL, CL, VH, and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15Rα Sushi domain; and the Fc region does not contain heterodimerization variant mutations. Figure ID (BFP4) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the modified IL-15 polypeptide comprising the L52C mutation comprises the amino acid sequence set forth in SEQ ID NO:22.

In some embodiments, the modified IL-15Rα Sushi domain comprising the S40C mutation comprises the amino acid sequence set forth in SEQ ID NO:23.

In some embodiments, the second polypeptide chain further comprises a second linker between the modified IL-15 polypeptide and the hinge region. In some embodiments, the second linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:33 or amino acids 21 to 610 of SEQ ID NO:33. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:32 The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), a CH1 domain, a first linker, a modified IL-15 polypeptide comprising the L52C mutation, a hinge region, and a modified Fc region; and (c) a modified IL-15RαSushi domain comprising the S40C mutation; wherein VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15RαSushi domain; and the modified Fc region contains one or more A mutation selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. Figure ID (BFP4) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the modified IL-15 polypeptide comprising the L52C mutation comprises the amino acid sequence set forth in SEQ ID NO:22.

In some embodiments, the modified IL-15Rα Sushi domain comprising the S40C mutation comprises the amino acid sequence set forth in SEQ ID NO:23.

In some embodiments, the second polypeptide chain further comprises a second linker between the modified IL-15 polypeptide and the hinge region. In some embodiments, the second linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the Fc region does not comprise heterodimerization variant mutations. In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:33 or amino acids 21 to 610 of SEQ ID NO:33. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:32 The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, IL-15 polypeptide, hinge region, and Fc region; and (c) IL-15RαSushi domain; wherein VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; The IL-15 polypeptide binds to the IL15Rα Sushi domain; and the Fc region does not contain heterodimerization variant mutations. Figure 1E (BFP5) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:20.

In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:21.

In some embodiments, the second polypeptide chain further comprises a second linker between the IL-15 polypeptide and the hinge region. In some embodiments, the second linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:8 or amino acids 21 to 610 of SEQ ID NO:8. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:7. The nucleic acid sequence shown encodes.

In another aspect, the disclosure herein provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain from N-terminus to C-terminus Contains: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) the second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH ), CH1 domain, first linker, IL-15 polypeptide, hinge region, and modified Fc region; and (c) IL-15RαSushi domain; wherein VL, CL, VH and CH1 together form an anti-PD-1 The Fab fragment; the IL-15 polypeptide binds to the IL15Rα Sushi domain; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. Figure 1E (BFP5) depicts an exemplary bifunctional homodimeric fusion protein according to this configuration.

In some embodiments, the VL comprises the light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, the light chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and the light chain of SEQ ID NO:6 Complementarity determining region 3 (LCDR3), and VH comprises heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO:2, and of SEQ ID NO:3 Heavy chain complementarity determining region 3 (HCDR3).

In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, and VH comprises the amino acid sequence of SEQ ID NO:13.

In some embodiments, the CL comprises an IgGl CK or Cλ domain. In some embodiments, CL comprises the amino acid sequence shown in SEQ ID NO:12.

In some embodiments, the CH1 domain comprises an IgG1 CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence shown in SEQ ID NO:11.

In some embodiments, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, VH and CH1 of the second polypeptide chain together comprise the amino acid sequence shown in SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35.

In some embodiments, the first linker comprises the amino acid sequence shown in SEQ ID NO:24.

In some embodiments, the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:20.

In some embodiments, the IL-15Rα Sushi domain comprises the amino acid sequence shown in SEQ ID NO:21.

In some embodiments, the second polypeptide chain further comprises a second linker between the IL-15 polypeptide and the hinge region. In some embodiments, the second linker comprises the amino acid sequence shown in SEQ ID NO:25.

In some embodiments, the hinge region is an IgG1 hinge region comprising the amino acid sequence shown in SEQ ID NO:17.

In some embodiments, the Fc region does not comprise heterodimerization variant mutations. In some embodiments, the heterodimerization variant mutations are selected from steric mutations, knob-hole mutations, electrostatic steering mutations, pi mutations, or any combination thereof. In some embodiments, the Fc region of each monomer does not comprise a paired amino acid substitution set selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/ E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T/E360E/Q362E:D401K；L368D/ K370S: S364K/E357L; D221E/P228E/L368E: D221R/P228R/K409R; C220E/P228E/368E: C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C: T366W/S354C.

In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, Δ236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19.

In some specific embodiments of this bifunctional homodimeric fusion protein, the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:34; And the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence shown in SEQ ID NO:8 or amino acids 21 to 610 of SEQ ID NO:8. In some specific embodiments of this bifunctional homodimer fusion protein, the first polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence shown in SEQ ID NO:7. The nucleic acid sequence shown encodes.

The polypeptide chains of the fusion proteins disclosed herein are not covalently bound to each other and can be interacted via ligand-ligand interactions (e.g., between the IL-15 polypeptide and the IL-15RαSushi domain) and disulfide bonds (e.g., with two VH-VL and hinge region of the polypeptide chain) self-assembly. VII , nucleic acid / vector / host cell

In another aspect, disclosed herein provides an isolated nucleic acid encoding a bifunctional homodimeric fusion protein comprising an anti-PD-1 binding domain component described herein and IL-15/IL-15Rα components. In some embodiments, the isolated nucleic acid encodes the first polypeptide chain, the second polypeptide chain, or both the first and second polypeptide chains of BFP1 or BFP6. In some embodiments, the isolated nucleic acid encodes the first polypeptide chain, the second polypeptide chain, or both the first and second polypeptide chains of BFP2, BFP3, BFP4, or BFP5. In a further embodiment, the co-expressed IL-15 polypeptide or IL-15Rα Sushi domain is on the same isolated nucleic acid as the first polypeptide chain and/or the second polypeptide chain of BFP2, BFP3, BFP4 or BFP5 encoded on, or encoded on an isolated nucleic acid different from the first polypeptide chain and/or the second polypeptide chain. In some embodiments, the first polypeptide chain, the second polypeptide chain, and optionally the IL-15 polypeptide or IL-15Rα Sushi domain are all encoded by a single nucleic acid.

The disclosure herein also provides isolated nucleic acid compositions comprising one or more nucleic acids encoding the bifunctional homodimeric fusion proteins described herein.

In some embodiments, the nucleic acid encoding a bifunctional homodimeric fusion protein comprising an anti-PD-1 binding domain component and an IL-15/IL-15Rα component is codon-optimized to enhance or Maximize performance in certain cell types (eg Scholten et al., Clin. Immunol . 119: 135–145, 2006). As used herein, a "codon-optimized" polynucleotide is a heterologous polypeptide having codons modified by silent mutations corresponding to the abundance of host cell tRNA concentrations.

In some embodiments, the nucleic acid molecule encoding the bifunctional homodimer fusion protein disclosed herein comprising an anti-PD-1 binding domain component and an IL-15/IL-15Rα component is self-cleaved by 2A A peptide separates the different polypeptide chains (eg, first and second polypeptide chains) contained therein. In some embodiments, the 2A self-cleaving peptide is Porcine Squad Virus-1 (P2A), Equine Rhinitis A Virus (E2A), Rhinoplasty Virus (T2A), Foot-and-Mouth Disease Virus (F2A), or any Combinations (see eg Kim et al.: PLOS One 6:e18556, 2011, the 2A nucleic acid and amino acid sequences of which are hereby incorporated by reference in their entirety).

In another aspect, an expression construct comprising a nucleic acid encoding a bifunctional homodimeric fusion protein as described herein is provided. In some embodiments, a nucleic acid can be operably linked to an expression control sequence (eg, an expression construct). As used herein, "expression construct" refers to a DNA construct comprising a nucleic acid molecule operably linked to suitable control sequences capable of effecting expression of the nucleic acid molecule in a suitable host. The expression construct can be present in a vector (eg bacterial vector, viral vector) or can be integrated into the genome. The term "operably linked" refers to the association of two or more nucleic acids on a single polynucleotide fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of affecting the expression of the coding sequence (ie, the coding sequence is under the transcriptional control of the promoter). The term "expression control sequence" (also referred to as a regulatory sequence) relates to a nucleic acid sequence which effects the expression and processing of a coding sequence to which it is operably linked. For example, expression control sequences may include transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that may enhance protein secretion.

In some embodiments, the nucleic acid or expression construct encoding the bifunctional homodimeric fusion protein is present in a vector. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector may, for example, be a plastid, cohesoplastid, virus, RNA vector, or linear or circular DNA or RNA molecule, which may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. Exemplary vectors are those capable of autonomous replication (episomal vectors) or expression of nucleic acid to which they are linked (expression vectors). Exemplary viral vectors include retroviruses, adenoviruses, parvoviruses (e.g. adeno-associated virus), coronaviruses, negative-sense RNA viruses such as orthomyxoviruses (e.g. influenza virus), rhabdoviruses (e.g. rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-sense RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, EB virus (Epstein-Barr virus, cytomegalovirus), and poxviruses (such as vaccinia, fowlpox, and canarypox). Other viruses include, for example, norovirus, chlamydiavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian type C, type B, type D, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, edited by B. N. Fields et al., Lippincott-Raven Publishers, Philadelphia, 1996). In some embodiments, the carrier is a plastid. In some other embodiments, the vector is a viral vector. In some such embodiments, the viral vector is a lentiviral vector or a gamma-retroviral vector.

The present disclosure also provides expression vector compositions comprising: a first expression vector comprising a first nucleic acid encoding a first polypeptide; a second expression vector comprising a second nucleic acid encoding a second polypeptide; and any Optionally, a third expression vector comprising a third nucleic acid encoding IL-15 polypeptide or IL-15RαSushi domain.

In another aspect, the disclosure herein also provides isolated host cells comprising nucleic acids (and nucleic acid compositions), expression constructs or vectors (vector combinations) encoding bifunctional homodimeric fusion proteins as described herein. things). As used herein, the term "host" refers to a cell or microorganism genetically modified with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (eg, IL-15 polypeptide). In certain embodiments, host cells may optionally already possess or be modified to include other genetic modifications that confer desired properties, whether related or not related to the biosynthesis of heterologous or exogenous proteins (e.g., including selectable marker). More than one heterologous or exogenous nucleic acid molecule can be introduced into the host cell as a single nucleic acid molecule, as multiple separately controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof . When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two or more exogenous acid molecules can be present as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, Integrate into the host chromosome at a single site or at multiple sites. References to the number of heterologous nucleic acid molecules or protein activities refer to the number of encoding nucleic acid molecules or protein activities, not the number of individual nucleic acid molecules introduced into the host cell.

Examples of host cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In some specific embodiments, the host cells are human embryonic kidney (HEK293) cells, YO cells, Sp2/0 cells, NSO mouse myeloma cells, PER.C6® human cells, baby hamster kidney cells (BHK), COS cells, or Chinese hamster ovary (CHO) cells. Host cells are cultured using methods known in the art.

In some embodiments, the disclosure herein provides an isolated host cell comprising: a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:34 and an amino acid encoding the amino acid having SEQ ID NO:27 sequence or the polynucleotide sequence of the polypeptide from amino acid 21 to 695 of SEQ ID NO:27; the polynucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:34 and encoding the polypeptide having the amino acid sequence of SEQ ID NO:10 Amino acid sequence or the polynucleotide sequence of the polypeptide of amino acids 21 to 695 of SEQ ID NO:10; the polynucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:34, encoding the polypeptide having SEQ ID NO The amino acid sequence of: 31 or the polynucleotide sequence of the polypeptide of amino acid 21 to 561 of SEQ ID NO: 31, and the polynucleotide sequence of the polypeptide of coding having the amino acid sequence of SEQ ID NO: 22; Encoding A polynucleotide sequence of a polypeptide having the amino acid sequence of SEQ ID NO:34, a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:33 or amino acids 21 to 610 of SEQ ID NO:33 sequence, and a polynucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO:23; encoding a polynucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO:34, encoding a polypeptide having an amino acid sequence of SEQ ID NO:8 The amino acid sequence of or the polynucleotide sequence of the polypeptide of amino acid 21 to 610 of SEQ ID NO:8, and the polynucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:21; A polynucleotide sequence of a polypeptide having an amino acid sequence of ID NO:34, a polynucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO:29 or amino acids 21 to 561 of SEQ ID NO:29, And the polynucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:20; or the polynucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:34 and encoding the polypeptide having the amino acid sequence of SEQ ID NO:54 An amino acid sequence or polynucleotide sequence of a polypeptide from amino acids 21 to 695 of SEQ ID NO:54; wherein the cell is capable of expressing a bifunctional homodimeric fusion protein.

In yet another aspect, disclosed herein provides a method of making a bifunctional homodimeric fusion protein as described herein, the method comprising culturing a host cell disclosed herein under suitable conditions for a period of time sufficient to express a bifunctional homodimeric fusion protein. The source dimeric fusion protein, and optionally isolating the bifunctional homodimeric fusion protein from the culture. Purification of bifunctional homodimeric fusion proteins can be performed according to methods known in the art. VIII , pharmaceutical composition

In another aspect, the disclosure herein provides a composition comprising the bifunctional homodimeric anti-PD1/IL-15/IL-15Rα fusion protein disclosed herein as described herein and a pharmaceutically acceptable carrier, diluted agents or excipients. Pharmaceutically acceptable carriers for diagnostic and therapeutic use are well known in the medical field and are described, for example, in Remington's Pharmaceutical Sciences , Mack Publishing Co. (Edited by AR Gennaro, 18th Edition, 1990) and the CRC Handbook of Food, Drug , and Cosmetic Excipients , CRC Press LLC (edited by SC Smolinski, 1992). Exemplary pharmaceutically acceptable carriers include any adjuvants, carriers, excipients, glidants, diluents, preservatives, dyes/colorants, surfactants, wetting agents, dispersing agents, suspending agents, stabilizing agents, agent, isotonic agent, solvent, emulsifier, or any combination thereof. For example, sterile saline at physiological pH and phosphate buffered saline may be suitable pharmaceutically acceptable carriers. Preservatives, stabilizers, dyes, or the like can also be provided in the pharmaceutical composition. In addition, antioxidants and suspending agents may be used. Pharmaceutical compositions may also contain diluents such as water, buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (fewer than about 10 residues), proteins, amino acids, carbohydrates (e.g., glucose, sucrose, dextrins) , chelating agents (such as EDTA), glutathione, and other stabilizers, and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary diluents.

The pharmaceutical compositions described herein can be formulated for oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intrasternal, and intratumoral injection, or infusion techniques.

In some embodiments, the pharmaceutical compositions of the invention are formulated as a single dosage unit or in a form comprising a plurality of dosage units. Methods for the preparation of such dosage forms are known or will be apparent to those skilled in the art; see, eg, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).

Pharmaceutical compositions can be in solid, semi-solid or liquid form. Solid compositions may include powders and tablets. In some embodiments, the pharmaceutical compositions described herein are in lyophilized or powder form for reconstitution with a suitable vehicle (eg, sterile water) before use. In some embodiments, the pharmaceutical compositions described herein are suspensions, solutions or emulsions. IX . How to use

In another aspect, disclosed herein provides a method of stimulating an immune response in an individual comprising administering to a patient in need thereof an effective amount of a bifunctional homodimeric fusion protein disclosed herein, or a bifunctional homodimeric fusion protein comprising a bifunctional homodimeric fusion protein disclosed herein. Pharmaceutical compositions derived from dimer fusion proteins.

In another aspect, disclosed herein provides a method for inhibiting tumor cells, comprising combining tumor cells with a bifunctional homodimeric fusion protein disclosed herein or a pharmaceutical combination comprising a bifunctional homodimeric fusion protein disclosed herein object contact.

The bifunctional homodimeric fusion protein disclosed herein can be used in a method of treating cancer, the method comprising administering to a patient in need thereof an effective amount of the bifunctional homodimeric fusion protein disclosed herein, or comprising the A pharmaceutical composition of a bifunctional homodimeric fusion protein.

Patients or individuals that may be treated by the bifunctional homodimeric fusion proteins disclosed herein include, but are not limited to, mammals, such as humans or non-human primates (e.g., monkeys and apes), domesticated animals (e.g., experimental animals, domestic pets or domestic animals), non-domesticated animals (such as wild animals), dogs, cats, rodents, mice, hamsters, cows, birds, chickens, fish, pigs, horses, goats, sheep, rabbits, and any combination thereof. In some embodiments, the individual is human. A subject can be male or female, and can be of any suitable age, including infant, juvenile, adolescent, adult, and elderly.

Medicine that "cures", "treats" or "improves" a disease, disorder or condition involving a subject, such as a human or non-human mammal such as a primate, horse, cat, dog, goat, mouse, or rat manage. Generally, a suitable dose or treatment regimen comprising a bifunctional homodimeric fusion protein or composition disclosed herein is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Treatment or prophylaxis/prevention benefit includes improved clinical outcome; lessening or palliation of disease-related symptoms; reduced incidence of symptoms; improved quality of life; longer disease-free status; reduction in disease extent; stabilization of disease state; delay in disease progression; remission ; survival; prolonged survival; or any combination thereof.

A "therapeutically effective amount" or "effective amount" of a bifunctional homodimeric fusion protein or composition disclosed herein refers to an amount of the molecule or composition sufficient to produce a therapeutic effect, including improved clinical outcomes; Associated symptoms; decreased incidence of symptoms; improved quality of life; longer disease-free state; lessened extent of disease; stabilization of disease state; delay in disease progression; remission; survival; or prolonged survival in a statistically significant manner. When referring to the administration of a single active ingredient alone, a therapeutically effective amount relates to the effect of that ingredient or the cells expressing that ingredient alone. When referring to a combination, a therapeutically effective amount relates to combined amounts of the active ingredients which produce a therapeutic effect, whether administered sequentially, sequentially or simultaneously. The combination can include, for example, a bifunctional homodimeric fusion protein and an antineoplastic agent.

The suitable dose, suitable duration and frequency of administration of the bifunctional homodimeric fusion protein or composition will be determined by factors such as the patient's condition, size, weight, body surface area, age, sex, type and severity of the disease, the condition to be administered, The particular therapy, the particular form of the active ingredient, the time and method of administration, and other drugs administered concomitantly depend on such factors as can be readily determined by those skilled in the art.

Generally, the therapeutically effective daily dose (for a 70 kg mammal) of the bifunctional homodimeric fusion protein is about 0.001 mg/kg (ie 0.07 mg) to about 100 mg/kg (ie 7.0 g); preferably Preferably, the therapeutically effective dose (for a 70 kg mammal) is about 0.01 mg/kg (ie 0.7 mg) to about 50 mg/kg (ie 3.5 g); more preferably, the therapeutically effective dose (for a 70 kg mammal) is About 1 mg/kg (ie 70 mg) to about 25 mg/kg (ie 1.75 g).

The bifunctional homodimeric fusion protein can be administered one or more times over a given period of time. In some embodiments, the method comprises administering the bifunctional homodimeric fusion protein to the individual at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.

In certain embodiments, the method comprises multiple administrations of the bifunctional homodimeric fusion protein to the individual, wherein the second or successive administrations are about 28 days, 21 days, 14 days, 10 days after the first administration , 7 days, 3 days, 1 day, or a shorter period of time.

The bifunctional homodimeric fusion proteins disclosed herein can be administered to an individual via the parenteral route. In some embodiments, the bifunctional homodimeric fusion protein is administered by subcutaneous, intravenous, intraarterial, subdural, intramuscular, intracranial, intrasternal, intratumoral, intraperitoneal, or infusion techniques into individual.

Cancers that can be treated by the bifunctional homodimeric fusion proteins disclosed herein include hematological malignancies and solid tumors. In some embodiments, the hematological malignancy is leukemia, lymphoma or myeloma. In some embodiments, the leukemia is acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute mononuclear leukemia, hairy cell leukemia, B-cell prolymphocytic leukemia, T cell Prolymphoblastic leukemia, or juvenile myelomonocytic leukemia. In some embodiments, the lymphoma is Hodgkin's lymphoma; non-Hodgkin's lymphoma; Epstein-Barr virus-associated lymphoproliferative disease; Burkitt lymphoma; large B-cell lymphoma, n.o.c. Description; Diffuse large B-cell lymphoma associated with chronic inflammation; Fibrin-associated diffuse large cell lymphoma; Primary effusion lymphoma; Plasmablastic lymphoma; Extranodal NK/T-cell lymphoma; Nasal type; peripheral T-cell lymphoma, n.o.c.; angioimmunoblastic T-cell lymphoma; follicular T-cell lymphoma; or systemic T-cell lymphoma in children. In some embodiments, the myeloma is multiple myeloma or myelodysplastic syndrome.

In some embodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, thymus carcinoma, malignant mesothelioma, pituitary tumor, thyroid tumor, melanoma tumor, Merkel cell skin cancer, lung cancer, head and neck cancer, colorectal cancer, liver cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, esophagus cancer, stomach cancer, small intestine cancer, anal cancer, kidney cancer, bladder cancer, prostate cancer, penis cancer , testicular cancer, breast cancer, ovarian cancer, cervical cancer, vaginal cancer, vulvar cancer, endometrial cancer, eye cancer, soft tissue sarcoma, hepatocellular carcinoma, brain tumor, or spinal cord tumor.

In some embodiments, the bifunctional homodimeric fusion proteins described herein can be used in combination with one or more antineoplastic agents. In some embodiments, the one or more antineoplastic agents are administered simultaneously, separately or sequentially. In some embodiments, the anti-tumor agent is cellular immunotherapy, antibody therapy, immune checkpoint inhibitor therapy, hormone therapy, chemotherapy, targeted cancer therapy, cytokine therapy, or any combination thereof. In some embodiments, cellular immunotherapy comprises TCR-T cell therapy, dendritic cell therapy or chimeric antigen receptor (CAR)-T cell therapy, or any combination thereof. In some embodiments, the antibody therapy comprises a promotive immune enhancing antibody. In some embodiments, antibody therapy comprises antibody-drug conjugates. In some embodiments, antibody therapy includes bevacizumab, nimotuzumab, lapatinib, cetuximab, panitumumab ), matuzumab, trastuzumab, nimotuzumab, zalutumumab, alemtuzumab, rituximab Anti-rituxmiab, magrolimab, or any combination thereof. In some embodiments, the immune checkpoint inhibitor therapy targets PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM- 3. CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, PVRIG/CD112R, CD47, SIRPα, or any combination thereof . In some embodiments, the immune checkpoint inhibitor therapy comprises ipilimumab, tremelimumab, pidilizumab, nivolumab, Pembrolizumab, durvalumab, atezolizumab, avelumab, urelumab, lirilumab ), or any combination thereof. In some embodiments, the hormone therapy comprises abiraterone, anastrozole, exemestane, fulvestrant, letrozole, leuprole Leuprolide, tamoxifen, or any combination thereof. In some embodiments, the cytokine therapy comprises IFNα, IL-2, IFNγ, GM-CSF, IL-7, IL-12, IL-21, or any combination thereof.

In some embodiments, chemotherapeutic agents comprise alkylating agents, platinum-based agents, cytotoxic agents, inhibitors of chromatin function, topoisomerase inhibitors, microtubule inhibitory drugs, DNA damaging agents, antimetabolites (such as folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interactors (such as intercalators), DNA repair inhibitors, or apoptosis inducers. Examples of chemotherapeutic agents considered for combination therapy include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (bleomycin) (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), Capecitabine (Xeloda®), N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU® ), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), arabinose Cytidine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), actinomycin d (dactinomycin) (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel ) (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil® , Efudex®), flutamide (Eulexin®), tezacitibine (tezacitibine), gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idamycin (Idamycin ®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), formyltetrahydrofolate, melphalan ( al keran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg (mylotarg), paclitaxel (Taxol®), Phoenix (yttrium 90/MX-DTPA), pentostatin, polyphenylene 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex® ), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin® ), vinblastine (Velban®), vincristine (Oncovin®) and vinorelbine (Navelbine®).

Exemplary alkylating agents include nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan® , Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), bischloroethylmethylamine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, RevimmuneTM), Ifosfamide (Mitoxana®), Melphalan (Alkeran®), Chlorambucil (Leukeran®), Pipobromide (Amedel®, Vercyte®), Ethylmelamine (Hemel®, Hexalen®, Hexastat®), Trisethylenethionylphosfosamide, Temozolomide (Temodar®), Thiotepa (Thioplex®), Busulfan (Busilvex®, Myleran®), Carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®) additional exemplary alkylating agents Including but not limited to Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Actinomycin d (also known as Actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin and melphalan, Alkeran®); hexamethylmelamine (also known as hexamethylmelamine (HMM), Hexalen®); carmustine (BiCNU®); bendamole Bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU, CeeNU®); chlorambucil (Leukeran®); cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome®); Altretamine (also known as Hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Pridemustine (P rednumustine); Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine, and mechlorethamine hydrochloride, Mustargen®); streptourea Streptozocin (Zanosar®); Thiotepa (also known as Thiophosphamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®) and bendamustine HCl (Treanda®).

Exemplary platinum-based agents include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthplatin ( phenanthriplatin), and triplatin tetranitrate (Triplatin tetranitrate).

Exemplary apoptosis inducers include AMG-224, AMG-176, and AMG-232, and venetoclax.

Exemplary targeted cancer therapies targeting specific molecules (e.g. oncogenes) involved in tumor growth, progression and metastasis, including angiogenesis inhibitors (e.g. VEGF pathway inhibitors), tyrosine kinase inhibitors (e.g. EGF pathway inhibitors) inhibitors), receptor tyrosine kinase inhibitors, growth factor inhibitors, GTPase inhibitors, serine/threonine kinase inhibitors, transcription factor inhibitors, B-Raf inhibitors, RAF inhibitors, MEK inhibitors Agents, mTOR inhibitors, EGFR inhibitors, ALK inhibitors, ROS1 inhibitors, BCL-2 inhibitors, PI3K inhibitors, VEGFR inhibitors, BCR-ABL inhibitors, MET inhibitors, MYC inhibitors, ABL inhibitors, HER2 inhibitors, BTK inhibitors, H-RAS inhibitors, K-RAS inhibitors, PDGFR inhibitors, TRK inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4/6 inhibitors, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PARP inhibitors, PDGFRA inhibitors, and RET inhibitors. In some embodiments, targeted cancer therapy includes bevacizumab, figitumumab, ramucirumab, ranibizumab, vemurafenib (vemurafenib), dabrafenib, encorafenib, vorinostat, binitinib, cobimetinib, rufametinib ( refametinib), selumetinib, trametinib, ibrutinib, tirabrutinib, acalabrutinib, secretinib ( spebrutinib), entrectinib, larotrectinib, lestaurtinib, imatinib, sunitinib, ponatinib, Capmatinib, crizotinib, tivantinib, onartuzumab, savolitinib, tepotinib, Papo Palbociclib, ribociclib, abemaciclib, trilaciclib, defactinib, erdafitinib, pemitinib ( pemigatinib, infigratinib, rogaratinib, quizartinib, crenolanib, gilteritinib, midostaurin , Lestaurtinib, Ivosidenib, Enasidenib, Talazoparib, Niraparib, Rucaparib, Oxygen Olaparib, veliparib, regorafenib, crenolanib, olaratumab, belvarafenib, Leva Lenvatinib, alectinib titinib), vandetanib, cabozantinib, ceritinib, lorlatinib, entrectinib, crizotinib, Ceritinib, Brigatinib, Osimeritinib, Icotinib, Gefitinib, Erlotinib, Abi proper (erbitux), or any combination thereof.

In another aspect, the bifunctional homodimer fusion proteins disclosed herein can be used in a method of treating a viral infection comprising administering to a patient in need thereof an effective amount of the bifunctional homodimer disclosed herein A fusion protein, or a pharmaceutical composition comprising the bifunctional homodimeric fusion protein disclosed herein. In some embodiments, the viral infection is an acute or chronic viral infection. The ability of the bifunctional homodimeric fusion proteins disclosed herein to activate T cells will be useful in the treatment of chronic infections. Infectious viruses include eukaryotic viruses such as adenoviruses, collapsarviruses, herpesviruses, papillomaviruses (e.g., HPV), paramyxoviruses, picornaviruses, rhabdoviruses (e.g., rabies), Myxoviruses (eg, influenza), poxviruses (eg, vaccinia), Rioviruses, retroviruses, lentiviruses (eg, HIV), flaviviruses (eg, HCV, HBV), coronaviruses, etc.

In some embodiments, the viral infection treated with the bifunctional homodimeric fusion proteins disclosed herein is caused by HIV, hepatitis (A, B or C), herpes virus (e.g. VZV, HSV-1, HAV-6 , HSV-II and CMV, EB virus), adenovirus, influenza virus, flavivirus, ECHO virus, rhinovirus, Coxsackie virus, coronavirus, respiratory fusion cell virus, mumps rubulavirus, rotavirus , measles morbillivirus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, or arbovirus encephalitis virus.

In some embodiments, the bifunctional homodimeric fusion proteins described herein are used in combination with one or more antiviral agents for the treatment of viral infections. In some embodiments, one or more antiviral agents are administered simultaneously, separately or sequentially. Example Materials and Methods Construction and Expression of Anti- PD1xIL15 Bifunctional Homodimer Fusion Protein

Construction of a series of bifunctional homodimeric fusion proteins (BFPs) comprising anti-PD-1 antibody components and IL-15 components and the Sushi domain of IL-15Rα (Sushi) (see Figures 1A to 1F) . The DNA sequence was cloned into a mammalian expression vector with human IgG1 Fc containing E233P, L234A, L235A, Δ236, A327G, A330S and P331S mutations to eliminate the effector functions (ADCC and CDC) of the Fc mediator. The relevant DNA sequence of IL-15 and/or Sushi domain is inserted into the heavy chain (H chain) of the IgG1 region between the Fab and the hinge. In some cases, DNA sequences for IL-15 or Sushi domains were inserted into mammalian expression vectors without an IgGl backbone for co-expression purposes. Figures 1A to 1F show schematics of these constructs. H-chain and L-chain vectors were co-expressed in Chinese hamster ovary (CHO) cells. In some cases, expression vectors of H chain, L chain and IL-15 or Sushi domain were co-expressed in CHO cells. Each of these polypeptide chains of the bifunctional homodimeric fusion protein can also be integrated into a single expression vector for the production of the fusion protein. Transfected CHO cells were incubated for 96 hours. The supernatant was then collected and purified using a protein A affinity column. ELISA binding assay

An enzyme-binding immunosorbent assay (ELISA) was used to assess whether the anti-PD-1 antibody component of the bifunctional homodimeric fusion protein retained its ability to bind human and macaque PD-1 proteins. 96-well plates were coated with 25 ng of recombinant human or Cynomolgus PD-1-Fc protein overnight at 4°C. The wells were blocked with PBS containing 1% BSA for 1 hour and washed with PBS containing 0.1% Tween-20. Then add serially diluted bifunctional fusion protein, anti-PD-1 antibody or control IgG (100 ul), and incubate at room temperature for 2 hours. After washing, the plates were incubated with HRP-conjugated anti-human IgG Fab antibody for 1 hour at room temperature. The plates are rinsed and then incubated with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine. Absorbance at 450 nm was measured on a microplate reader. The percent binding relative to control IgG was calculated. Flow cytometric analysis of PD-1 transfected Jurkat cells by anti- PD-1 antibody and bifunctional homodimeric fusion protein

Jurkat cells transfected with human PD-1 were stained with anti-PD1xIL15 fusion protein, anti-PD-1 antibody or isotype control IgG1 followed by BB700-conjugated anti-human Fab antibody. Cells were then analyzed by flow cytometry. The mean fluorescence units (MFU) were plotted against the concentration of the protein reagent. PD-1 luciferase reporter assay

A pair of engineered cell lines were used in this assay: 1) PD-1 ⁺ effector cells (GloResponse NFAT-luc2/PD1 Jurkat cells), which are PD-1 expressing and luciferase driven by NFAT response elements Reporter Jurkat T cells, and 2) PD-L1 ⁺ antigen-presenting cells (PD-L1 aAPC/CHO-K1 cells), which are CHO-K1 cells expressing PD-L1 and engineered cell surface TCR activating proteins. When the two cell types were co-cultured, the PD-1/PD-L1 interaction reduced TCR signaling and NFAT-mediated luminescence. Addition of reagents containing an antagonist anti-PD-1 antibody that blocks PD-1/PD-L1 interaction abolished co-inhibitory signaling and resulted in TCR activation and enhanced luminescence of NFAT-RE mediators.

PD-L1 aAPC/CHO-K1 human T-activated cells (Promega) were seeded at 40,000 cells/well in 96-well white opaque dishes in 100 μl of RPMI-1640 medium containing 10% FBS at 37 °C, 5% CO ₂ and incubated overnight. The medium was removed from the assay plate the next day, and various concentrations of anti-PD1xIL15 bifunctional homodimer fusion protein, anti-PD-1 antibody, control antibody, or control IL15RαSushi-IL-15 were added in 40 μl buffer per well - Fc fusion protein. GloResponse NFAT-luc2/PD1 Jurkat cells (Promega) ^were resuspended in assay buffer at 1.25 x 106/ml and added to the plate at 40 μl per well. After 6 hours of incubation, the assay plates were allowed to equilibrate for 5 minutes at room temperature. Bio-Glo™ reagent (Promega) was added to each well at 80 μl per well. The plates were then incubated at room temperature for 5 minutes. Luminescence was measured in a plate reader. Mixed Leukocyte Reaction ( MLR ) Assay

CD4 ⁺ T cells were isolated from healthy donors using the EasySep Human CD4 ⁺ T Cell Isolation Kit (Stemcell Technologies, Vancouver, BC). Peripheral blood mononuclear cells (PBMC) (Stemcell Technologies) from another healthy donor were incubated in RPMI1640 medium with 10% fetal bovine serum for 3 hours at 37°C, 5% CO ₂ . Non-adherent cells were removed and the remaining adherent cells were used for dendritic cell production by culturing for 4 days in RPMI1640 medium containing 10% fetal bovine serum containing IL-4 (20 ng /mL) and granulocyte-macrophage colony-stimulating factor (20 ng/mL, R&D systems).

For the MLR assay, CD4 ⁺ T cells were mixed with allogeneic DC at a ratio of 10:1 in AIM-V medium containing 0.5% BSA. This was followed by addition of bifunctional homodimeric anti-PD1xIL15 fusion protein, PD-1 antibody or IgG controls at serially diluted concentrations ranging from 133.25 nM to 1.33 pM. After 4 days of culture, supernatants were collected and IFN-γ production was measured by ELISA assay. Flow cytometric analysis of NK cell activation

Human peripheral NK cells isolated from PBMCs of healthy donors were cultured for 24 hours in the presence or absence of 20 nM bifunctional aPD1xIL15 fusion protein, PD-1 antibody, or IL15RSushi-IL15 fusion protein control. Triple staining was performed with CD25-APC/CD56-PE/CD69-FITC. After washing, cells were analyzed for NK cell activation markers by flow cytometry. CD56 ⁺ NK cells were analyzed against CD25 or CD69. Measuring the release of IFN-γ or granzyme B to K526 target cells in an NK cytotoxicity assay

Human peripheral NK cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. It was incubated for 48 hours in the presence of IL-2. NK cells were co-cultured with K562 cells for 4 hours in the presence or absence of bifunctional homodimeric anti-PD1xIL15 fusion protein, PD-1 antibody, or IL15RSushi-IL15 fusion protein controls. Release of IFN-γ or granzyme B in co-culture supernatants was measured by ELISA. MTS assay to measure T cell proliferation

The MTS assay is a colorimetric method for the quantification of viable cells. Human anti-CD3 activated T cells were maintained by 10 ng/ml IL-2. After washing, cells were treated with bifunctional homodimeric anti-PD1xIL15 fusion protein, control antibody or IL-15 for 72 hours. The MTS tetrazolium compound was then added to the cell culture medium and the plates were incubated for 2 hours. Measure the absorbance at 490 nm for T cell proliferation. Example 1 : Construction and expression of anti- PD1xIL15 bifunctional homologous fusion protein

Create an anti-PD1xIL15 bifunctional fusion protein design (see Figures 1A to 1G). Abbreviations are as follows: VL=light chain variable region; CL=light chain constant region; VH=heavy chain variable region; CH1=heavy chain constant region 1; Sushi=Sushi domain of IL-15 receptor alpha; hinge=IgG1 hinge region; and Fc=fragment crystallizable region.

By pairing the VL-CL sequence (SEQ ID NO:34) of the anti-PD-1 IgG1 antibody with its VH-CH1 sequence linked to the Sushi sequence and IL-15 sequence, followed by the hinge-Fc sequence (SEQ ID NO:27 ) paired to construct BFP1 (Fab-Sushi-IL-15-Fc) (Figure 1A).

BFP2 (Fab-Sushi-Fc/co-IL15) was constructed by co-expression of IL-15 and Sushi-containing IgG1-like structures. This containing sequence was constructed by pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgG1 antibody with the heavy chain VH-CH1 sequence linked to the Sushi-hinge-Fc sequence (SEQ ID NO:29). IgG1-like structure of Sushi (Fig. 1B). BFP3 (Fab-Sushi(mu)-Fc/co-IL15(mu)) was constructed by co-expression of IL-15 containing point mutation (L52C) and IgG1-like structure containing Sushi. By pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgG1 antibody with the heavy chain VH-CH1 sequence linked to Sushi containing a point mutation (S40C), followed by the hinge-Fc sequence (SEQ ID NO:34) NO:31) to construct the IgG1-like structure containing Sushi (Fig. 1C).

BFP4 (Fab-IL15(mu)-Fc/co-Sushi(mu)) was constructed by co-expression of Sushi containing point mutation (S40C) and IgG1-like structure containing IL15. By pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgG1 antibody with the heavy chain VH-CH1 sequence linked to IL-15 containing a point mutation (L52C), followed by the hinge-Fc sequence ( SEQ ID NO:33) to construct the IL15-containing IgG1-like structure (Fig. 1D).

BFP5 (Fab-IL15-Fc/co-Sushi) was constructed by co-expression of Sushi with IgG1-like structures containing IL-15. By pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgG1 antibody with the heavy chain VH-CH1 sequence linked to IL-15, followed by pairing with the hinge-Fc sequence (SEQ ID NO:8) , and construct this IgG1-like structure containing IL15 (Fig. 1E).

By pairing the VL-CL sequence (SEQ ID NO:34) of the anti-PD-1 IgG1 antibody with its VH-CH1 sequence linked to the IL-15 sequence and the Sushi sequence, followed by the hinge-Fc sequence (SEQ ID NO:10 ) paired to construct BFP6 (Fab-IL-15-Sushi-Fc) (Figure 1F).

By pairing the VL-CL sequence (SEQ ID NO:34) of the anti-PD-1 IgG1 antibody with its VH-CH1 sequence linked to the Sushi sequence and IL-15 I67E sequence, followed by the hinge-Fc sequence (SEQ ID NO: 54) paired to construct BFP7 (Figure 16). These BFP1, BFP2, BFP3, BFP4 and BFP7 constructs were used to transfect Chinese hamster cells (CHO) for transient or stable expression. The supernatants of these expressed cultures were purified using the Protein A method. Endotoxin concentrations were determined to be less than 0.2 units per mg protein. Example 2 : In Vitro Functional Study Anti- PD1xIL15 Bifunctional Homodimer Fusion Protein Retains Binding Activity to Recombinant Human or Cynomolgus Monkey PD-1

96-well plates were coated with 25 ng of recombinant human or macaque PD-1-Fc protein. Serial dilutions of bifunctional homodimeric fusion protein or control antibody were added. Bound bifunctional homodimeric fusion protein or antibody is detected using an HRP-conjugated anti-human IgG Fab antibody and a chromogenic substrate at OD450 nm. All of these bifunctional homodimeric anti-PD1xIL15 fusion proteins did not differ significantly in their ability to bind human or cynomolgus recombinant PD-1 Fc protein compared to their parental anti-PD-1 monoclonal antibodies (Fig. 2B). Anti- PD1xIL15 bifunctional homodimeric fusion protein can simultaneously bind PD1 and IL-15Rβγ

The ability of bifunctional homodimeric anti-PD1xIL15 fusion proteins (BFP1 to BFP4) to simultaneously bind two targets (PD-1 and IL-15Rβγ) was analyzed by double binding ELISA. The results showed that these BFPs could simultaneously bind PD-1 and IL-15Rβγ. A 96-well plate was coated with 25 ng per well of recombinant human PD-1-Fc protein. Add serially diluted antibody or fusion protein. After washing, bound antibody or fusion protein was detected with 5 ng per well of biotinylated IL15Rβγ and streptavidin-HRP, followed by chromogenic TMB matrix. The anti-PD-1 antibody and IL15 components in the fusion protein can simultaneously bind PD-1 protein and IL15R/β (Figure 3). Anti- PD1xIL15 bifunctional homodimeric fusion protein retains binding activity to cell surface PD-1

The ability of the bifunctional homodimeric anti-PD1xIL15 fusion protein to bind to a cell line expressing human PD-1 was then tested. Jurkat cells transfected with human PD-1 were stained with anti-PD1xIL15 fusion protein, anti-PD-1 antibody or isotype control IgG1 followed by BB700-conjugated anti-human Fab antibody. Cells were then analyzed by flow cytometry. The mean fluorescence units (MFU) were plotted against the concentration of the protein reagent. Among the different bifunctional fusion proteins, there was no significant difference in their binding activity to cell surface PD-1 (Figure 4). Anti- PD1xIL15 bifunctional homodimeric fusion protein retains the ability to enhance T cell activation in a PD-1 luciferase reporter assay

The ability of the bifunctional anti-PD1xIL15 fusion protein to enhance T cell activation was compared to that of the parental anti-PD-1 antibody in a PD-1 luciferase reporter assay. PD-L1 aAPC/CHO-K1 human T-activator in the presence or absence of anti-PD-1 antibody, bifunctional homodimeric fusion protein, control IgG, or control IL15RαSushi-IL15 fusion protein Cells were co-cultured with GloResponse NFAT-luc2/PD-1 Jurkat cells for 6 hours. Luminescent activity was measured in a plate reader (Figure 5). An anti- PD1xIL15 bifunctional homodimeric fusion protein retains the ability to enhance T cell activation in a mixed leukocyte reaction ( MLR ) assay

The ability of the bifunctional homodimeric anti-PD1xIL15 fusion protein to enhance T cell activation in the MLR assay was also investigated. Similar to the PD-1 antibody, treatment with the bifunctional anti-PD1xIL15 fusion protein significantly enhanced T cell activation as measured by IFN-γ release (Figure 6). In contrast, IL-15 itself or the IL-15/IL5RαSushi complex had no effect on T cell activation. Effects of bifunctional homodimeric anti- PD1xIL15 fusion protein on NK cell activation

Natural killer (NK) cells are part of the innate immune system and mediate responses against tumor cells. The ability of the bifunctional homodimeric anti-PD1xIL15 fusion protein, the control IL15RαSushi-IL15-Fc fusion protein, or the control antibody to activate CD56 ⁺ NK cells was analyzed. CD25 and CD69 are considered as NK cell activation markers. Treatment with anti-PD1xIL15 bispecific homodimeric fusion protein significantly increased the number of activated NK cells by CD25 or CD69 expression assay, similar to IL15RαSushi-IL15-Fc fusion protein (Fig. 7A-7B). In contrast, treatment with anti-PD-1 antibody or an isotype control IgG1 had no effect. Effect of bifunctional anti- PD1xIL15 fusion protein on NK cytotoxic activity

Whether the bifunctional homodimeric anti-PD1xIL15 fusion protein enhances NK cytotoxic activity by IFN-γ and granzyme B production was analyzed. Human peripheral NK cells were isolated and cultured for 48 hours in the presence of IL-2. K562 is a sensitive target cell line for NK cells. NK cells were co-cultured with K562 cells for 4 hours in the presence or absence of bifunctional homodimeric anti-PD1xIL15 fusion protein, anti-PD-1 antibody, or IL15RSushi-IL15-Fc fusion protein controls. There was no effect in anti-PD-1 antibody or control IgG1. IFN-γ and granzyme B release were shown in co-culture supernatants of NK cells cultured in different types of bifunctional homodimeric anti-PD1xIL15 fusion proteins other than BFP4, IL15RS, Sushi-IL15 fusion proteins, or IL15 significantly increased, suggesting that the bifunctional homodimeric anti-PD1xIL15 fusion protein retains the ability of IL15 to enhance NK cell-mediated cytotoxic activity (Figure 8). In repeated studies, similar results were observed with dose-response effects (data not shown).

NK activation can also be measured by secretion of granzyme B from NK cells in a cytotoxicity assay against K562 target cells. Granzyme was observed in co-culture supernatants of NK cells and K562 target cells treated with different types of bifunctional homodimeric anti-PD1xIL15 fusion proteins other than BFP4, IL15RSushi-IL15 fusion protein or IL15 The significant increase in B release suggested that the bifunctional aPD1xIL15 fusion protein retained the ability of the IL15 component to enhance NK cell-mediated cytotoxic activity (Fig. 9). In repeated studies, similar results were observed with dose-response effects (data not shown). Bifunctional anti- PD1xIL15 bifunctional homodimeric fusion protein promotes T cell proliferation

IL-15 plays an important role in the proliferation and survival of T cells (especially memory T effector cells). The ability of the IL15 component of the bifunctional homodimeric anti-PD1xIL15 fusion protein to promote T cell proliferation was tested in the MTS assay. Anti-CD3 antibody-activated T cells were cultured for 72 h in the presence of bifunctional homodimeric anti-PD1xIL15 protein, control antibody, or IL15RαSushi-IL15 complex. The results showed that all bifunctional reagents retained the ability of the IL15 complex to promote T cell proliferation to varying degrees (Figure 10). In contrast, anti-PD-1 antibodies had little effect on T cell proliferation. Example 3 : In vivo functional studies of mouse tumor models

Human PD-1 knock-in syngeneic mice were inoculated with 1 x10 ⁵ MC38 tumor cells. When tumor size reached approximately 120 mm, mice were treated with anti-PD1xIL15 bifunctional homodimeric fusion proteins ^BFP1 , BFP2, parental anti-PD1 antibody, or human IgG at indicated doses and intervals. Anti-PD1 antibody #61 significantly slowed tumor growth compared to human IgG (control) treatment. Treatment with BFP1 or BFP2 appeared to have better anti-tumor efficacy than anti-PD1 antibody #61 alone (Figure 11). Example 4 : Stability analysis of bifunctional homodimer anti- PD1xIL15 fusion protein

The bifunctional homodimeric fusion proteins BFP1 to BFP4 were treated at 4°C, room temperature and 37°C for 1 week, 2 weeks or 4 weeks. The processed fusion protein was then diluted and loaded into 96-well plates coated with 25 ng/well of recombinant human PD-1-Fc protein. After removal of unbound fusion protein, bound fusion protein was detected using HRP-conjugated anti-human IgG Fab antibody and a chromogenic matrix at OD450 nm. These temperature-treated BFPs remained stable as measured by PD-1 binding activity ( FIGS. 12A to 12D ). PD-L1 aAPC/CHO-K1 human T-activated somatic cells react with GloResponse NFAT-luc2/PD-1 Jurkat in the presence or absence of bifunctional homodimeric fusion proteins BFP1 to BFP4 or control IgG Cells were co-cultured for 6 hours. Luminescent activity was measured in a plate reader. These temperature-treated BFPs remained stable as measured by their ability to enhance T cell activation (Figure 13). Human peripheral NK cells were isolated and cultured for 48 hours in the presence of IL-2. NK cells were co-cultured with K562 cells for 4 hours in the presence or absence of temperature-treated bifunctional homodimeric fusion proteins BFP1 to BFP4. IFN-γ released in co-culture supernatants was measured by ELISA. These temperature-treated BFPs remained functionally stable as measured by NK cell function assays (Figures 14A to 14D).

The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patent applications, and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including U.S. patent applications filed on March 9, 2021 No. 63/158,801, both of which are incorporated herein by reference in their entirety. Aspects of the specific embodiments can be modified, if desired, to employ concepts of the various patents, applications and documents to provide other specific embodiments. These and other changes can be made to the particular embodiments in light of the above detailed description. In general, in the following scope of claims, the words used should not be construed as limiting the scope of claims to specific embodiments disclosed in the specification and scope of claims, but should be interpreted as including all possible specific embodiments and The full scope of equivalents to which such claims are entitled. Therefore, the patent scope of the application is not limited by the disclosure herein.

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Figures 1A to 1G show exemplary schematic diagrams of anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins ("BFPs"): (Figure 1A) "BFP1"; (Figure 1B) "BFP2"; (Figure 1B) 1C) "BFP3"; (Fig. 1D) "BFP4"; (Fig. 1E) "BFP5"; (Fig. 1F) "BFP6"; and (Fig. 1G) "BFP7". Figures 2A to 2B show the results of an ELISA binding assay demonstrating that the anti-PD-1 binding domain in the bifunctional homodimeric fusion protein retains the ability to bind human (Figure 2A) and Cynomolgus monkey (Figure 2B) PD-1 proteins . Figure 3 shows PD1 and IL15Rβ/γ double binding ELISA. Figure 4 shows flow cytometric analysis of PD-1 transfected Jurkat cells stained with anti-PD-1 antibody or various anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins. Figure 5 shows the enhancement of T cell activation by various anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins in a PD-1 luciferase reporter assay. Figure 6 shows that T cell activation is enhanced by various anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins in an MLR assay. Figures 7A to 7B show flow cytometric analysis of human peripheral NK cells treated with anti-PD-1 antibodies, IL-15 fusion proteins, or various anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins, using Expressed on CD69 (Figure 7A) and CD25 (Figure 7B). Figure 8 shows IFN in human peripheral NK cells co-cultured with K562 cells after treatment with anti-PD-1 antibody, IL-15 fusion protein, or various anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins - Gamma secretion assay. Figure 9 shows the expression of granzyme B in NK cells co-cultured with K562 cells after treatment with anti-PD-1 antibodies, IL-15 fusion proteins, or various anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins Secretion analysis. Figure 10 shows the MTS assay to measure the effects of anti-PD-1 antibodies, IL-15 fusion proteins, or various anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins on T cell proliferation. Figure 11 shows that human-PD-1 knock-in (knock- in) Tumor volume in the MC3 mouse tumor model. Figures 12A - 12D show stability assays for exemplary anti-PD-1 x IL-15 bifunctional homodimeric fusion proteins ("BFPs"). Temperature-treated BFPs were used in PD-1 binding ELISA assays (Figure 12A - BFP1, Figure 12B - BFP2, Figure 12C - BFP3, Figure 12D - BFP4). Figure 13 shows the results of a stability test of temperature-treated BFP in a PD-1 luciferase reporter assay to measure its ability to enhance T cell activation. Figures 14A to 14D show the results of stability testing of temperature-treated BFPs (Figure 14A - BFP1, Figure 14B - BFP2, Figure 14C - BFP3, Figure 14D - BFP4) in NK cell function assays.

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Claims (57)

A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, IL-15RαSushi domain, second linker, IL-15 polypeptide, hinge region, and Fc region; Wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment, and the Fc region does not contain heterodimerization variant mutations. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) The second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, IL-15 polypeptide, second linker, IL - 15RαSushi domain, hinge region, and Fc region; Wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment, and the Fc region does not contain heterodimerization variant mutations. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, modified IL-15RαSushi structure containing S40C mutation domain, hinge region, and Fc region; and (c) a modified IL-15 polypeptide comprising the L52C mutation; wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15Rα Sushi domain; and the Fc region does not contain a heterodisulfide bond Merging variant mutations. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, modified IL-15 polypeptide comprising L52C mutation , a hinge region, and an Fc region; and (c) Modified IL-15Rα Sushi domain containing the S40C mutation; wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15Rα Sushi domain; and the Fc region does not contain a heterodisulfide bond Merging variant mutations. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, IL-15 polypeptide, hinge region, and Fc region ;as well as (c) IL-15RαSushi domain; Wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the IL-15 polypeptide binds to the IL15Rα Sushi domain; and the Fc region does not contain a heterodimerization variant mutation. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, IL-15RαSushi domain, second linker, IL-15 polypeptide, hinge region, and modified Fc region; Wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); and (b) The second polypeptide chain, which comprises from N-terminal to C-terminal: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, IL-15 polypeptide, second linker, IL -15RαSushi domain, hinge region, and modified Fc region; Wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, IL-15RαSushi domain, hinge region, and via a modified Fc region; and (c) IL-15 polypeptides; Wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the IL-15 polypeptide binds to the IL15RαSushi domain; and the modified Fc region comprises one or more selected from E233P, L234V, L235A, Δ236 , A327G, A330S, and P331S mutations. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, IL-15 polypeptide, hinge region, and modified the Fc region of; and (c) IL-15RαSushi domain; Wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the IL-15 polypeptide binds to the IL15RαSushi domain; and the modified Fc region comprises one or more selected from E233P, L234V, L235A, Δ236 , A327G, A330S, and P331S mutations. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, modified IL-15RαSushi structure containing S40C mutation domain, hinge region, and modified Fc region; and (c) a modified IL-15 polypeptide comprising the L52C mutation; wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15RαSushi domain; and the modified Fc region comprises One or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. A bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) the first polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody light chain variable region (VL) and light chain constant region (CL); (b) The second polypeptide chain, which comprises from N-terminus to C-terminus: anti-PD1 antibody heavy chain variable region (VH), CH1 domain, first linker, modified IL-15 polypeptide comprising L52C mutation , a hinge region, and a modified Fc region; and (c) Modified IL-15Rα Sushi domain containing the S40C mutation; wherein the VL, CL, VH and CH1 together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide forms a covalent disulfide bond with the modified IL-15RαSushi domain; and the modified Fc region comprises One or more mutations selected from E233P, L234V, L235A, Δ236, A327G, A330S, and P331S. The bifunctional homodimeric fusion protein according to any one of claims 1 to 5, wherein the heterodimerization variant mutation is selected from space mutation, knob (knob)-hole (hole) mutation, electrostatic steering mutation, pi mutation, or any combination thereof. The bifunctional homodimeric fusion protein according to claim 12, wherein the Fc region of each monomer does not include a paired amino acid substitution group selected from: S364K/E357Q:L368D/K370S; L368D/K370S:S364K ；L368E/K370S:S364K；T411T/E360E/Q362E:D401K；L368D/K370S:S364K/E357L；K370S:S364K/E357Q；T366S/L368A/Y407V:T366W；S267K/L368D/K370S:S267K/LS364K/E357Q；T411T /E360E/Q362E:D401K; L368D/K370S:S364K/E357L; D221E/P228E/L368E:D221R/P228R/K409R; C220E/P228E/368E:C220R/E224R/K409R; S354C. The bifunctional homodimeric fusion protein according to any one of claims 1 to 13, wherein the anti-PD-1 Fab fragment formed by the VH and CH1 of the first polypeptide and the second polypeptide is chimeric , human or humanized. The bifunctional homodimer fusion protein according to any one of claims 1 to 14, wherein the anti-PD-1 Fab fragment formed by the VH and CH1 of the first polypeptide and the second polypeptide is IgG1, IgG2, IgG3, or IgG4 isotype. The bifunctional homodimeric fusion protein according to any one of claims 1 to 15, wherein the VL comprises light chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, light chain complementarity of SEQ ID NO:5 Determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3) of SEQ ID NO: 6, and the VH includes heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 1, of SEQ ID NO: 2 Heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO:3. The bifunctional homodimer fusion protein according to any one of claims 1 to 16, wherein the VL comprises the amino acid sequence of SEQ ID NO: 14, and the VH comprises the amino acid sequence of SEQ ID NO: 13 . The bifunctional homodimer fusion protein according to any one of claims 1 to 17, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34, and the VH and CH1 comprises the amino acid sequence of SEQ ID NO:35 or amino acids 21 to 236 of SEQ ID NO:35. The bifunctional homodimer fusion protein according to any one of claims 1 to 18, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 24 (SGGSGGGGSGGGSGGGGSLQ). The bifunctional homodimer fusion protein according to any one of claims 1, 2, 6 or 7, wherein the second linker comprises the amino acid sequence of SEQ ID NO: 24 (SGGSGGGGSGGGSGGGGSLQ). The bifunctional homodimer fusion protein according to any one of claims 1, 2 and 5 to 9, wherein the IL-15RαSushi domain comprises the amino acid sequence of SEQ ID NO:21. The bifunctional homodimer fusion protein according to any one of claims 1, 2 and 5 to 9, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:20. The bifunctional homodimer fusion protein according to claim 6, wherein the IL-15 polypeptide comprises a modified IL-15 polypeptide, and the modified IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:52. The bifunctional homodimer fusion protein according to any one of claims 3, 4, 10 and 11, wherein the modified IL-15RαSushi domain comprises the amino acid sequence of SEQ ID NO:23. The bifunctional homodimer fusion protein according to any one of claims 3, 4, 10 and 11, wherein the modified IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:22. The bifunctional homodimeric fusion protein according to any one of claims 1 to 25, wherein the hinge region is an IgG1, IgG2, IgG3 or IgG4 hinge region. The bifunctional homodimeric fusion protein according to any one of claims 1 to 26, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 17 (PKSCDKTHTCPPCPAPPVAGP). The bifunctional homodimeric fusion protein according to claim 1 or 6, wherein the second polypeptide chain further comprises a third linker between the IL-15 polypeptide and the hinge region. The bifunctional homodimer fusion protein according to any one of claims 4, 5, 9 and 11, wherein the second polypeptide chain further comprises a second connection between the IL-15 polypeptide and the hinge region son. The bifunctional homodimeric fusion protein according to claim 2 or 7, wherein the second polypeptide chain further comprises a third linker between the IL-15RαSushi domain and the hinge region. The bifunctional homodimer fusion protein according to any one of claims 3, 8 and 10, wherein the second polypeptide chain further comprises a second linker between the IL-15RαSushi domain and the hinge region . The bifunctional homodimeric fusion protein according to claim 28 or 30, wherein the third linker comprises the amino acid sequence of SEQ ID NO: 25 (GGGSIEGRMD). The bifunctional homodimeric fusion protein according to claim 29 or 31, wherein the second linker comprises the amino acid sequence of SEQ ID NO: 25 (GGGSIEGRMD). Such as the bifunctional homodimeric fusion protein of claim 1, wherein: (a) the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO: 34; and (b) The second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO:27 or amino acids 21 to 695 of SEQ ID NO:27. Such as the bifunctional homodimeric fusion protein of claim 2, wherein: (a) the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO: 34; and (b) The second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO:10 or amino acids 21 to 695 of SEQ ID NO:10. Such as the bifunctional homodimeric fusion protein of claim 3, wherein: (a) the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO:34; (b) the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO: 31 or amino acids 21 to 561 of SEQ ID NO: 31; and (c) The modified IL-15 polypeptide of each monomer comprises the amino acid sequence of SEQ ID NO:22. Such as the bifunctional homodimer fusion protein of claim 4, wherein: (a) the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO:34; (b) the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO: 33 or amino acids 21 to 610 of SEQ ID NO: 33; and (c) The modified IL-15RαSushi domain comprises the amino acid sequence of SEQ ID NO:23. Such as the bifunctional homodimeric fusion protein of claim 5, wherein: (a) the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO:34; (b) the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO: 8 or amino acids 21 to 610 of SEQ ID NO: 8; and (c) The IL-15RαSushi domain comprises the amino acid sequence of SEQ ID NO:21. Such as the bifunctional homodimeric fusion protein of claim 6, wherein: (a) the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO:34; (b) the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO: 29 or amino acids 21 to 561 of SEQ ID NO: 29; and (c) The IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:20. Such as the bifunctional homodimeric fusion protein of claim 6, wherein: (a) the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO:34; (b) The second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence of SEQ ID NO:54 or amino acids 21 to 695 of SEQ ID NO:54. A pharmaceutical composition comprising the bifunctional homodimer fusion protein according to any one of claims 1 to 40 and a pharmaceutically acceptable carrier. An isolated nucleic acid composition encoding the bifunctional homodimer fusion protein according to any one of claims 1-40. An isolated nucleic acid composition comprising: (a) a first nucleic acid encoding the first polypeptide chain according to any one of claims 1 to 40; (b) a second nucleic acid encoding a second polypeptide chain according to any one of claims 1 to 40; and optionally, (c) a third nucleic acid encoding the modified IL-15 polypeptide according to claim 3 or 10, the modified IL-15RαSushi domain according to claim 4 or 11, the IL-15RαSushi domain according to claim 5 or 9 domain, or the IL-15 polypeptide according to claim 8. An expression vector comprising the nucleic acid composition according to claim 42 or 43. A performance carrier composition comprising: (a) a first expression vector comprising a first nucleic acid encoding the first polypeptide according to any one of claims 1 to 40; (b) a second expression vector comprising a second nucleic acid encoding a second polypeptide according to any one of claims 1 to 40; and optionally, (c) a third expression vector comprising a third nucleic acid encoding the modified IL-15 polypeptide according to claim 3 or 10, the modified IL-15Rα Sushi domain according to claim 4 or 11, The IL-15Rα Sushi domain according to claim 5 or 9, or the IL-15 polypeptide according to claim 8. An isolated host cell comprising the nucleic acid composition according to claim 42 or 43, the expression vector according to claim 44, or the expression vector composition according to claim 45. An isolated host cell expressing the bifunctional homodimeric fusion protein according to any one of claims 1 to 40. An isolated host cell comprising: (a) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:34 and a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:27 or amino acids 21 to 695 of SEQ ID NO:27; (b) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:34 and a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:10 or amino acids 21 to 695 of SEQ ID NO:10; (c) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:34, a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:31 or amino acids 21 to 561 of SEQ ID NO:31, And the nucleic acid encoding the polypeptide having the amino acid sequence of SEQ ID NO:22; (d) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:34, a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:33 or amino acids 21 to 610 of SEQ ID NO:33, And the nucleic acid encoding the polypeptide having the amino acid sequence of SEQ ID NO:23; (e) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:34, a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:8 or amino acids 21 to 610 of SEQ ID NO:8, And the nucleic acid encoding the polypeptide having the amino acid sequence of SEQ ID NO:21; (f) a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:34, a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:29 or amino acids 21 to 561 of SEQ ID NO:29, And the nucleic acid encoding the polypeptide having the amino acid sequence of SEQ ID NO:20; (g) A nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:34 and a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO:54 or amino acids 21 to 695 of SEQ ID NO:54. A method for producing the bifunctional homodimeric fusion protein according to any one of claims 1 to 40, which comprises culturing such as claimed items 46 to 48 under conditions suitable for expressing the bifunctional homodimeric fusion protein Any one of the host cells. The method according to claim 49, further comprising isolating the bifunctional homodimeric fusion protein. A method for inhibiting tumor cells, comprising contacting the tumor cells with the bifunctional homodimeric fusion protein according to any one of claims 1 to 40 or the pharmaceutical composition according to claim 41. A method of stimulating an immune response in an individual, comprising administering the bifunctional homodimeric fusion protein according to any one of claims 1 to 40 or the pharmaceutical composition according to claim 41 to the individual. A method of treating cancer in an individual, comprising administering to the individual an effective amount of the bifunctional homodimeric fusion protein according to any one of claims 1 to 40 or the pharmaceutical composition according to claim 41. The method according to claim 53, wherein the cancer is a hematological malignancy or a solid tumor. The method of claim 54, wherein the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, thymus carcinoma, malignant mesothelioma, pituitary tumor, thyroid tumor , melanoma, Merkel cell skin cancer, lung cancer, head and neck cancer, colorectal cancer, liver cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, esophagus cancer, stomach cancer, small intestine cancer, anal cancer, kidney cancer, bladder cancer, prostate cancer, Cancer of the penis, testicle, breast, ovary, cervix, vagina, vulva, endometrium, eye, soft tissue sarcoma, or hepatocellular carcinoma, brain tumor, or spinal cord tumor. A method for treating viral infection in an individual, comprising administering to the individual an effective amount of the bifunctional homodimeric fusion protein according to any one of claims 1 to 40 or the pharmaceutical composition according to claim 39. The method of any one of claims 53 to 56, further comprising simultaneously, separately or sequentially administering one or more additional therapeutic agents.

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